Inbred corn line kw4er1502

ABSTRACT

Inbred corn line, designated KW4ER1502, are disclosed. The disclosure relates to the seeds of inbred corn line KW4ER1502, to the plants and plant parts of inbred corn line KW4ER1502 and to methods for producing a corn plant, either inbred or hybrid, by crossing inbred corn line KW4ER1502 with itself or another corn line. The disclosure also relates to products produced from the seeds, plants, or parts thereof, of inbred corn line KW4ER1502 and/or of the hybrids produced using the inbred as a parent. The disclosure further relates to methods for producing a corn plant containing in its genetic material one or more transgenes and to the transgenic plants produced by that method and to methods for producing other corn lines derived from inbred corn line KW4ER1502.

FIELD

The present disclosure relates to a new and distinctive corn inbred line(Zea mays, also known as maize), designated KW4ER1502.

BACKGROUND

Corn is an important and valuable field crop. Thus, a continuing goal ofplant breeders is to develop stable, high yielding corn hybrids that areagronomically sound. The reasons for this goal are obviously to maximizethe amount of ears or kernels produced on the land used and to supplyfood for both humans and animals. To accomplish this goal, the cornbreeder must select and develop corn plants that have the traits thatresult in superior parental lines for producing hybrids.

SUMMARY OF THE DISCLOSURE

The following embodiments and aspects thereof are described inconjunction with systems, tools and methods which are meant to beexemplary and illustrative, not limiting in scope. In variousembodiments, one or more of the above-described problems have beenreduced or eliminated, while other embodiments are directed to otherimprovements.

According to the disclosure, provided is a novel inbred corn linedesignated KW4ER1502. This disclosure thus relates to seeds of inbredcorn line KW4ER1502, to plants or parts thereof of inbred corn lineKW4ER1502, to plants or parts thereof having all the physiological andmorphological characteristics of inbred corn line KW4ER1502 and toplants or parts thereof having all the physiological and morphologicalcharacteristics of inbred corn line KW4ER1502 listed in Table 1,including but not limited to as determined at the 5% significance levelwhen grown in the same environmental conditions. In some embodiments, arepresentative sample of seed of the inbred conr line is deposited.

The disclosure also relates to variants, mutants and trivialmodifications of the seed or plant of inbred corn line KW4ER1502. Partsof the inbred corn plant of the present disclosure are also provided,such as e.g., pollen obtained from an inbred plant and an ovule of theinbred plant. Variants, mutants and trivial modifications of the seed orplant of the corn line of the present disclosure can be generated bymethods available to one skilled in the art, including but not limitedto, mutagenesis (e.g., chemical mutagenesis, radiation mutagenesis,transposon mutagenesis, insertional mutagenesis, signature taggedmutagenesis, site-directed mutagenesis, and natural mutagenesis),knock-outs/knock-ins, antisense and RNA interference. For moreinformation of mutagenesis in plants, such as agents, protocols, seeAcquaah et al. (Principles of plant genetics and breeding,Wiley-Blackwell, 2007, ISBN 1405136464, 9781405136464, which is hereinincorporated by reference in its entity).

As used herein, the verb “comprise” as is used in this description andin the claims and its conjugations are used in its non-limiting sense tomean that items following the word are included, but items notspecifically mentioned are not excluded.

The term “a” or “an” refers to one or more of that entity; for example,“a gene” refers to one or more genes or at least one gene. As such, theterms “a” (or “an”), “one or more” and “at least one” are usedinterchangeably herein. In addition, reference to “an element” by theindefinite article “a” or “an” does not exclude the possibility thatmore than one of the elements are present, unless the context clearlyrequires that there is one and only one of the elements.

The disclosure also relates to a mutagenized population of inbred cornline KW4ER1502, and methods of using such populations. In someembodiments, the mutagenized population can be used in screening for newcorn lines which comprises one or more or all of the morphological andphysiological characteristics of inbred corn line KW4ER1502. In someembodiments, the new corn lines obtained from the screening processcomprise all the morphological and physiological characteristics ofinbred corn line KW4ER1502, and one or more additional or differentmorphological and physiological characteristics that the inbred cornline KW4ER1502 does not have.

The mutagenized population of the present disclosure can be used inTargeting Induced Local Lesions in Genomes (TILLING) screening method,which combines a standard and efficient technique of mutagenesis with achemical mutagen (e.g., Ethyl methanesulfonate (EMS)) with a sensitiveDNA screening-technique that identifies single base mutations (alsocalled point mutations) in a target gene. Detailed description onmethods and compositions on TILLING® can be found in Till et al.(Discovery of induced point mutations in maize genes by TILLING, BMCPlant Biology 2004, 4:12), Weil et al., (TILLING in Grass Species, PlantPhysiology January 2009 vol. 149 no. 1 158-164), Comai, L. and S.Henikoff (“TILLING: practical single-nucleotide mutation discovery.”Plant J 45(4): 684-94), McCallum et al., (Nature Biotechnology, 18:455-457, 2000), McCallum et al., (Plant Physiology, 123: 439-442, 2000),Colbert et al., (Plant Physiol. 126(2): 480-484, 2001), U.S. Pat. No.5,994,075, U.S. Patent Application Publication No. 2004/0053236A1, andInternational Patent Application Publication Nos. WO2005/055704 andWO2005/048692, each of which is hereby incorporated by reference for allpurposes.

The plants and seeds of the present disclosure include those that may beof an essentially derived variety as defined in section 41(3) of thePlant Variety Protection Act, i.e., a variety that:

-   -   1. is predominantly derived from inbred corn line KW4ER1502, or        from a variety that is predominantly derived from inbred corn        line KW4ER1502, while retaining the expression of the essential        characteristics that result from the genotype or combination of        genotypes of inbred corn line KW4ER1502;    -   2. is clearly distinguishable from inbred corn line KW4ER1502;        and    -   3. except for differences that result from the act of        derivation, conforms to the initial variety in the expression of        the essential characteristics that result from the genotype or        combination of genotypes of the initial variety.

In another aspect, the present disclosure provides regenerable cells foruse in tissue culture of inbred corn plant corn line KW4ER1502. Thetissue culture will be capable of regenerating plants having all thephysiological and morphological characteristics of the foregoing inbredcorn plant of the disclosure. Preferably, the cells of such tissuecultures will be embryos, ovules, meristematic cells, seeds, callus,pollen, leaves, anthers, roots, root tips, silk, flowers, kernels, ears,cobs, husks, stalks or the like. Protoplasts produced from such tissueculture are also included in the present disclosure. The corn shoots,roots and whole plants regenerated from the tissue cultures are alsopart of the disclosure.

Also included in this disclosure are methods for producing a corn plantproduced by crossing the inbred corn line KW4ER1502 with itself oranother corn line. When crossed with itself, i.e., crossed with anotherinbred corn line KW4ER1502 plant or self-pollinated, the inbred linecorn line KW4ER1502 will be conserved (e.g., as an inbred). When crossedwith another, different corn line, an F₁ hybrid seed is produced. F₁hybrid seeds and plants produced by growing said hybrid seeds areincluded in the present disclosure. A method for producing an F₁ hybridcorn seed comprising crossing inbred corn line KW4ER1502 corn plant witha different corn plant and harvesting the resultant hybrid corn seed arealso part of the disclosure. The hybrid corn seed produced by the methodcomprising crossing inbred corn line KW4ER1502 corn plant with adifferent corn plant and harvesting the resultant hybrid corn seed areincluded in the disclosure, as are included the hybrid corn plant orparts thereof, seeds included, produced by growing said hybrid cornseed.

In another embodiment, this disclosure relates to a method for producingthe inbred corn line KW4ER1502 from a collection of seeds, thecollection containing both inbred corn line KW4ER1502 seeds and hybridseeds having inbred corn line KW4ER1502 as a parental line. Such acollection of seeds might be a commercial bag of seeds. Said methodcomprises planting the collection of seeds. When planted, the collectionof seeds will produce inbred corn line KW4ER1502 plants from inbred cornline KW4ER1502 seeds and hybrid plants from hybrid seeds. The plantshaving all the physiological and morphological characteristics of inbredcorn line KW4ER1502 or having a decreased vigor compared to the otherplants grown from the collection of seeds are identified as inbred cornline KW4ER1502 parent plant. Said decreased vigor is due to theinbreeding depression effect and can be identified for example by a lessvigorous appearance for vegetative and/or reproductive characteristicsincluding shorter plant height, small ear size, ear and kernel shape,ear color or other characteristics. As previously mentioned, if theinbred corn line KW4ER1502 is self-pollinated, the inbred corn lineKW4ER1502 will be preserved, therefore, the next step is controllingpollination of the inbred parent plants in a manner which preserves thehomozygosity of said inbred corn line KW4ER1502 parent plant and thefinal step is to harvest the resultant seed.

This disclosure also relates to methods for producing other corn linesderived from inbred corn line KW4ER1502 and to the corn lines derived bythe use of those methods.

In another aspect, the present disclosure provides transformed inbredcorn line KW4ER1502 or parts thereof that have been transformed so thatits genetic material contains one or more transgenes, preferablyoperably linked to one or more regulatory elements. Also, the disclosureprovides methods for producing a corn plant containing in its geneticmaterial one or more transgenes, preferably operably linked to one ormore regulatory elements, by crossing transformed inbred corn lineKW4ER1502 with either a second plant of another corn line, or anon-transformed corn plant of the inbred corn line KW4ER1502, so thatthe genetic material of the progeny that results from the cross containsthe transgene(s), preferably operably linked to one or more regulatoryelements. The disclosure also provides methods for producing a cornplant that contains in its genetic material one or more transgene(s),wherein the method comprises crossing the inbred corn line KW4ER1502with a second plant of another corn line which contains one or moretransgene(s) operably linked to one or more regulatory element(s) sothat the genetic material of the progeny that results from the crosscontains the transgene(s) operably linked to one or more regulatoryelement(s). Transgenic corn plants, or parts thereof produced by themethod are in the scope of the present disclosure.

More specifically, the disclosure comprises methods for producing cornplants or seeds with at least one trait selected from the groupconsisting of male sterile, male fertile, herbicide resistant, insectresistant, disease resistant, water stress tolerant corn plants orseeds, or corn plants or seeds with modified, in particular decreased,phytate content, with modified waxy and/or amylose starch content, withmodified protein content, with modified oil content or profile, withincreased digestibility or with increased nutritional quality. Saidmethods comprise transforming the inbred corn line KW4ER1502 corn plantwith nucleic acid molecules and/or transgenes that confer, for example,male sterility, male fertility, herbicide resistance, insect resistance,disease resistance, water stress tolerance, or that can modify thephytate, the waxy and/or amylose starches, the protein or the oilcontents, the digestibility or the nutritional qualities, respectively.The transformed corn plants or seeds obtained from the provided methods,including, for example, those corn plants or seeds with male sterility,male fertility, herbicide resistance, insect resistance, diseaseresistance, water stress tolerance, modified phytate, waxy and/oramylose starches, protein or oil contents, increased digestibility andincreased nutritional quality are included in the present disclosure.Plants may display one or more of the above listed traits. For thepresent disclosure and the skilled artisan, disease is understood to befungal disease, viral disease, bacterial disease or other plantpathogenic diseases and disease resistant plant encompasses plantsresistant to fungal, viral, bacterial and other plant pathogens.

Also included in the disclosure are methods for producing a corn plantor seed containing in its genetic material one or more transgenesinvolved with fatty acid metabolism, carbohydrate metabolism, and starchcontent such as waxy starch or increased amylose starch. The transgeniccorn plants or seeds produced by these methods are also part of thedisclosure.

In another aspect, the present disclosure provides for methods ofintroducing one or more desired trait(s) into the inbred corn lineKW4ER1502 and plants or seeds obtained from such methods. The desiredtrait(s) may be, but not exclusively, a single gene, preferably adominant but also a recessive allele. Preferably, the transferred geneor genes will confer such traits as male sterility, herbicideresistance, insect resistance, resistance for bacterial, fungal, orviral disease, male fertility, water stress tolerance, enhancednutritional quality, modified waxy content, modified amylose content,modified protein content, modified oil content, enhanced plant quality,enhanced digestibility and industrial usage. The gene or genes may benaturally occurring gene(s), transgene(s), mutant(s), or genes modifiedthrough New Breeding Techniques. In some embodiments, the method forintroducing the desired trait(s) is a backcrossing process making use ofa series of backcrosses to the inbred corn line KW4ER1502 during whichthe desired trait(s) is maintained by selection. The single locusconversion plants that can be obtained by the method are included in thepresent disclosure.

In some embodiments, the backcross breeding process of inbred corn lineKW4ER1502 comprises (a) crossing inbred corn line KW4ER1502 with plantsthat comprise the desired trait(s) to produce F1 progeny plants. In someembodiments, the process further comprises (b) selecting the F1 progenyplants that have the desired trait(s); In some embodiments, the processfurther comprises (c) crossing the selected F1 progeny plants with theinbred corn line KW4ER1502 plants to produce backcross progeny plants;In some embodiments, the process further comprises (d) selecting forbackcross progeny plants that have the desired trait(s) and all thephysiological and morphological characteristics of the inbred corn lineKW4ER1502 to produce selected backcross progeny plants; In someembodiments, the process further comprises (e) repeating steps (c) and(d) one, two, three, four, five six, seven, eight, nine or more times insuccession to produce selected, second, third, fourth, fifth, sixth,seventh, eighth, ninth or higher backcross progeny plants that have thedesired trait(s) and consist essentially of the phenotypic andmorphological characteristics of the inbred corn line KW4ER1502, and/orhave all the phenotypic and morphological characteristics of the inbredcorn line KW4ER1502, and/or have the desired trait(s) and thephysiological and morphological characteristics of the inbred corn lineKW4ER1502 as determined in Table 1, including but not limited to at a 5%significance level when grown in the same environmental conditions. Thecorn plants or seeds produced by the method are also part of thedisclosure, as are the inbred corn line KW4ER1502 that comprise thedesired trait(s). Backcrossing breeding methods, well known to oneskilled in the art of plant breeding will be further developed insubsequent part of the specification.

When dealing with a gene that has been modified, for example through NewBreeding Techniques, the trait (genetic modification) could be directlymodified into the newly developed line/cultivar such as inbred corn lineKW4ER1502. Alternatively, if the trait is not modified into each newlydeveloped line/cultivar such as inbred corn line KW4ER1502, anothertypical method used by breeders of ordinary skill in the art toincorporate the modified gene is to take a line already carrying thegene and to use such line as a donor line to transfer the gene into thenewly developed line. The same would apply for a naturally occurringtrait or one arising from spontaneous or induced mutations.

When using a transgene, the trait is generally not incorporated intoeach newly developed line such as inbred corn line KW4ER1502 by directtransformation. Rather, the more typical method used by breeders ofordinary skill in the art to incorporate the transgene is to take a linealready carrying the transgene and to use such line as a donor line totransfer the transgene into the newly developed line. The same wouldapply for a naturally occurring trait (e.g., a native trait, such as butnot limited to drought tolerance or improved nitrogen utilization) orone arising from spontaneous or induced mutations. In some embodiments,the backcross breeding process comprises (a) crossing inbred corn lineKW4ER1502 plants with plants of another line that comprise the desiredtrait(s). In some embodiments, the process further comprises (b)selecting the F₁ progeny plants that have the desired trait(s). In someembodiments, the process further comprises (c) crossing the selected F₁progeny plants with the inbred corn line KW4ER1502 plants to producebackcross progeny plants. In some embodiments, the process furthercomprises (d) selecting for backcross progeny plants that have thedesired trait(s) and all the physiological and morphologicalcharacteristics of corn line KW4ER1502 to produce selected backcrossprogeny plants. In some embodiments, the process further comprises (e)repeating steps (c) and (d) one, two, three, four, five, six, seven,eight, nine or more times in succession to produce selected, second,third, fourth, fifth, sixth, seventh, eighth, ninth or higher backcrossprogeny plants that comprise the desired trait(s) and all thephysiological and morphological characteristics of corn line KW4ER1502as listed in Table 1, including but not limited to at a 5% significancelevel when grown in the same environmental conditions. The corn plantsor seeds produced by the methods are also part of the disclosure.Backcrossing breeding methods, well known to one skilled in the art ofplant breeding will be further developed in subsequent parts of thespecification.

In another aspect of the disclosure, The hybrid corn plants or seedshaving inbred corn line KW4ER1502 can be used as a parent that containsone or more transgenes, and may be crossed with another corn line. Inanother aspect of the disclosure, a single locus converted inbred lineor a transgenic inbred corn line of KW4ER1502 can be used as a parentand may be crossed with another corn line. Preferably, the single locusconverted inbred lines or transgenic inbred lines will confer suchtraits, herbicide resistance, insect resistance, resistance forbacterial, fungal, or viral disease, male fertility, water stresstolerance, enhanced nutritional quality, modified waxy content, modifiedamylose content, modified protein content, modified oil content,enhanced plant quality, enhanced digestibility and industrial usage. Thegene or genes may be naturally occurring maize gene(s) (e.g., nativetraits) or transgene(s) introduced through genetic engineeringtechniques. The hybrid corn plants or seeds having inbred corn lineKW4ER1502 or a single converted inbred corn line KW4ER1502 or atransgenic inbred corn line KW4ER1502 as a parental line and havinganother, different, corn line as a second parental line as discussedabove are comprised in the present disclosure.

Any DNA sequence(s), whether from a different species or from the samespecies that is inserted into the genome using transformation isreferred to herein collectively as “transgenes.” In some embodiments ofthe disclosure, a transformed variant of KW4ER1502 may contain at leastone transgene but could contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 transgenes. In another embodiment of the disclosure, a transformedvariant of another corn line used as the other parental line may containat least one transgene but could contain at least 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 transgenes, such as MON603, MON810, MON88017, MON89034,TC1507, T25 and DAS-59122-7.

In an embodiment of this disclosure is a method of making a backcrossconversion of inbred corn line KW4ER1502, comprising the steps ofcrossing a plant of corn inbred KW4ER1502 with a donor plant comprisinga mutant gene(s), a naturally occurring gene(s), or a gene(s) and/orsequences modified through the use of New Breeding Techniques,conferring one or more desired trait to produce F₁ progeny plant. Insome embodiments, the method comprises backcrossing the selected F₁progeny plant to a plant of inbred corn line KW4ER1502, or plantcomprising the naturally occurring gene(s), mutant gene(s) or modifiedgene(s) and/or sequence conferring the one or more desired trait. Thismethod may further comprise the step of obtaining a molecular markerprofile of inbred corn line KW4ER1502 and using the molecular markerprofile to select for a progeny plant with the desired trait and themolecular marker profile of inbred corn line KW4ER1502. In the samemanner, this method may be used to produce an F₁ hybrid seed by adding afinal step of crossing the desired trait conversion of inbred corn lineKW4ER1502 with a different corn plant to make F₁ hybrid corn seedcomprising a mutant gene or transgene conferring the desired trait.

In some embodiments of the disclosure, the number of loci that may bebackcrossed into inbred corn line KW4ER1502 is at least 1, 2, 3, 4, 5,or more. A single locus may contain several genes, such as a gene fordisease resistance, or a gene herbicide resistance. The gene forherbicide resistance may be used as a selectable marker and/or as aphenotypic trait. A single locus conversion of site specific integrationsystem allows for the integration of multiple genes at the convertedlocus. In some embodiments, a single locus conversion of site specificintegration system allows for the integration of multiple genes at theconverted locus. A single locus conversion also allows for making one ormore site specific changes to the plant genome, such as, withoutlimitation, one or more nucleotide changes, deletions, insertions, etc.In some embodiments, the single locus conversion is performed by genomeediting, a.k.a. genome editing with engineered nucleases (GEEN). In someembodiments, the genome editing comprises using one or more engineerednucleases. In some embodiments, the engineered nucleases include, butare not limited to Zinc finger nucleases (ZFNs), TranscriptionActivator-Like Effector Nucleases (TALENs), the CRISPR/Cas system (usingsuch as Cas9, Cas12a/Cpf1, Cas13/C2c2, CasX and CasY), and engineeredmeganuclease, engineered homing endonucleases and endonucleases for forDNA guided genome editing (Gao et al., Nature Biotechnology (2016), doi:10.1038/nbt.3547). In some embodiments, the single locus conversionchanges one or several nucleotides of the plant genome. Such genomeediting techniques are some of the techniques now known by a personskilled in the art and herein are collectively referred to as ‘NewBreeding Techniques’. In some embodiments, the single locus conversionchanges one or several nucleic acids of the plant genome. In someembodiments, the plant comprising a single locus conversion andotherwise essentially all of the phenotypical and morphologicalcharacteristics of an inbred plant of the present disclosure, such asall of the characteristics listed in Table 1 when grown under the sameenvironmental conditions.

In a preferred embodiment, the present disclosure provides methods forincreasing and producing inbred corn line KW4ER1502 seed, whether bycrossing a first inbred corn plant with a second inbred corn plant andharvesting the resultant corn seed, wherein both said first and secondinbred corn plant are the inbred corn line KW4ER1502 or by planting aninbred corn seed of the inbred corn line KW4ER1502, growing an inbredcorn line KW4ER1502 plant from said seed, controlling a self pollinationof the plant where the pollen produced by the grown inbred corn lineKW4ER1502 plant pollinates the ovules produced by the very same inbredcorn line KW4ER1502 grown plant and harvesting the resultant seed.

The disclosure further provides methods for developing corn plants in acorn plant breeding program using plant breeding techniques includingmutation breeding, wherein said mutation breeding selects for a mutationthat is spontaneous or artificially induced, recurrent selection,backcrossing, pedigree breeding, molecular marker enhanced selection(using Isozyme Electrophoresis, Restriction Fragment LengthPolymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs),Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA AmplificationFingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs).Amplified Fragment Length Polymorphisms (AFLPs), and Simple SequenceRepeats (SSRs) which are also referred to as Microsatellites, etc.),genetic marker enhanced selection, haploid/double haploid production,and transformation. Corn seeds, plants, and parts thereof produced bysuch breeding methods are also part of the disclosure.

In addition, any and all products made using the corn seeds, plants andparts thereof obtained from inbred corn line KW4ER1502 or from any cornline produced using inbred corn line KW4ER1502 as a direct or indirectparent are also part of the disclosure. Examples of such corn productsinclude but are not limited to corn meal, corn flour, corn starch, cornsyrup, corn sweetener and corn oil. The origin of the corn used in suchcorn products can be determined by tracking the source of the corn usedto make the products and/or by using protein (isozyme, ELISA, etc.)and/or DNA (RFLP, PCR, SSR, SNP, EST, etc.) testing.

The disclosure also relates to variants, mutants and trivialmodifications of the seed or plant of the inbred corn line KW4ER1502.Variants, mutants and trivial modifications of the seed or plant ofinbred corn line KW4ER1502 can be generated by methods available to oneskilled in the art, including but not limited to, mutagenesis (e.g.,chemical mutagenesis, radiation mutagenesis, transposon mutagenesis,insertional mutagenesis, signature tagged mutagenesis, site-directedmutagenesis, and natural mutagenesis), knock-outs/knock-ins, antisense,RNA interference and other techniques such as the New BreedingTechniques. For more information of mutagenesis in plants, such asagents, protocols, see Acquaah et al. (Principles of plant genetics andbreeding, Wiley-Blackwell, 2007, ISBN 1405136464, 9781405136464, whichis herein incorporated by reference in its entity).

The disclosure also relates to a mutagenized population of the inbredcorn line KW4ER1502 and methods of using such populations. In someembodiments, the mutagenized population can be used in screening for newcorn plants which comprises one or more or all of the morphological andphysiological characteristics of inbred corn line KW4ER1502. In someembodiments, the new corn plants obtained from the screening processcomprise all of the morphological and physiological characteristics ofthe inbred corn line KW4ER1502, and one or more additional or differentmorphological and physiological characteristics that inbred corn lineKW4ER1502 does not have.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by study of thefollowing descriptions.

Definitions

In the description and tables which follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Allele: The allele is any of one or more alternative forms of a gene,all of which relate to one trait or characteristic. In a diploid cell ororganism, the two alleles of a given gene occupy corresponding loci on apair of chromosomes. Two alleles of the genetic sequence in diploidcells correspond to the same locus (i.e., position) on homologouschromosomes.

Backcrossing: Backcrossing is a process in which a breeder repeatedlycrosses hybrid progeny back to one of the parents, for example, a firstgeneration hybrid F₁ with one of the parental genotype of the F₁ hybrid.

BT1-1, BT: BT1-1 refers to MON810, also known as MON810Bt or BT1, is thedesignation given by the Monsanto Company (St. Louis, MO) for thetransgenic event that, when expressed in maize, produces an endotoxinthat is efficacious against the European corn borer, Ostrinia nubilalisand certain other Lepidopteran larvae.

Collection of seeds: In the context of the present disclosure acollection of seeds will be a grouping of seeds mainly containingsimilar kind of seeds, for example hybrid seeds having the inbred lineof the disclosure as a parental line, but that may also contain, mixedtogether with this first kind of seeds, a second, different kind ofseeds, of one of the inbred parent lines, for example the inbred line ofthe present disclosure. A commercial bag of hybrid seeds having theinbred line of the disclosure as a parental line and containing also theinbred line seeds of the disclosure would be, for example such acollection of seeds.

CL: CL, also known as Clearfield is commercial denomination given to thegene that, when present in maize, allows the use of imidazolinoneherbicides as a weed control agent for both grasses (i.e.,monocotyledons) and broadleaves (i.e., dicotyledons).

Daily heat unit value: The daily heat unit value (also referred to asgrowing degree unit, or GDU) is calculated as follows: (the maximumdaily temperature+the minimum daily temperature)/2 minus 50. Alltemperatures are in degrees Fahrenheit. The maximum temperaturethreshold is 86 degrees, if temperatures exceed this, 86 is used. Theminimum temperature threshold is 50 degrees, if temperatures go belowthis, 50 is used. For each hybrid, it takes a certain number of GDUs toreach various stages of plant development. GDUs are a way of measuringplant maturity. GDUs can also relate to stages of growth for an inbredline.

Decreased vigor: A plant having a decreased vigor in the presentdisclosure is a plant that, compared to other plants has a less vigorousappearance for vegetative and/or reproductive characteristics includingshorter plant height, small ear size, ear and kernel shape, ear color orother characteristics.

Dropped ears: This is a measure of the number of dropped ears per plot,and represents the percentage of plants that dropped an ear prior toharvest.

Dry down: This is the rate at which a hybrid will reach acceptableharvest moisture.

Ear height. The ear height is a measure from the ground to the upper earnode attachment, and is measured in centimeters.

Essentially all of the physiological and morphological characteristics:A plant having essentially all of the physiological and morphologicalcharacteristics means a plant having all of the physiological andmorphological characteristics of a plant of the present disclosure,except for additional traits and/or mutations which do not materiallyaffect the plant of the present disclosure, or a desiredcharacteristic(s), which can be indirectly obtained from another plantpossessing at least one single locus conversion via a conventionalbreeding program (such as backcross breeding) or directly obtained byintroduction of at least one single locus conversion via New BreedingTechniques. In some embodiments, one of the non-limiting examples for aplant having (and/or comprising) essentially all of the physiologicaland morphological characteristics shall be a plant having all of thephysiological and morphological characteristics of a plant of thepresent disclosure other than desired, additionaltrait(s)/characteristic(s) conferred by a single locus conversionincluding, but not limited to, a converted or modified gene.

GDU pollen: The number of heat units from planting until 50% of theplants in the hybrid are shedding pollen.

GDU silk: The GDU silk (=heat unit silk) is the number of growing degreeunits (GDU) or heat units required for an inbred line or hybrid to reachsilk emergence from the time of planting.

Harvest aspect: This is a visual rating given the day of harvest or theprevious day. Hybrids are rated 1 (poorest) to 9 (best) with poorerscores given for poor plant health, visible signs of fungal infection,poor plant intactness characterized by missing leaves, tassels, or othervegetative parts, or a combination of these traits.

Herbicide resistant or tolerant: A plant containing anyherbicide-resistant gene or any DNA molecule or construct (or replicatethereof) which is not naturally occurring in the plant which results inincrease tolerance to any herbicide including, imidazoline,sulfonylurea, glyphosate, glufosinate, L-phosphinothricin, triazine andbenzonitrile. For purposes of this definition, a DNA molecule orconstruct shall be considered to be naturally occurring if it exists ina plant at a high enough frequency to provide herbicide resistancewithout further selection and/or if it has not been produced as a resultof tissue culture selection, mutagenesis, genetic engineering usingrecombinant DNA techniques or other in vitro or in vivo modification tothe plant.

Inbreeding depression: The inbreeding depression is the loss ofperformance of the inbreds due to the effect of inbreeding, i.e., due tothe mating of relatives or to self-pollination. It increases thehomozygous recessive alleles leading to plants which are weaker andsmaller and having other less desirable traits.

Introduce or Introducing: Introducing in the context of a plant cell,plant and/or plant part means contacting a nucleic acid molecule withthe plant, plant part, and/or plant cell in such a manner that thenucleic acid molecule gains access to the interior of the plant celland/or a cell of the plant and/or plant part (i.e. transformation). Italso refers to both the natural and artificial transmission of a desiredallele, transgene, or combination of desired alleles of a genetic locusor genetic loci, or combination of desired transgenes from one geneticbackground to another. For example, a desired allele or transgene at aspecified locus can be transmitted to at least one progeny via a sexualcross between two parents of the same species, where at least one of theparents has the desired allele or transgene in its genome.Alternatively, for example, transmission of an allele or transgene canoccur by recombination between two donor genomes, e.g., in a fusedprotoplast, where at least one of the donor protoplasts has the desiredallele in its genome. The desired allele may be a selected allele of amarker, a QTL, a transgene, or the like. Offspring comprising thedesired allele or transgene can be repeatedly backcrossed to a linehaving a desired genetic background and selected for the desired alleleor transgene, with the result being that the desired allele or transgenebecomes fixed in the desired genetic background. In some embodiments,transgene(s) and/or a single locus conversion can be introduced throughgenetic engineering techniques including the New Breedign Techniquestaught in the disclosure. In other embodiments, a vector and/or plasmidcan be introduced into a cell, a tissue, an organ, or an organism bytransformation and/or transfection.

Late plant greenness: Similar to a stay green rating. This is a visualassessment given at around the dent stage but typically a few weeksbefore harvest to characterize the degree of greenness left in theleaves. Plants are rated from 1 (poorest) to 9 (best) with poorer scoresgiven for plants that have more non-green leaf tissue typically due toearly senescence or from disease.

Locus: A locus is a defined segment of DNA. This segment is oftenassociated with an allele position on a chromosome.

MN RM: This represents the Minnesota Relative Maturity Rating (MN RM)for the hybrid and is based on the harvest moisture of the grainrelative to a standard set of checks of previously determined MN RMrating. Regression analysis is used to compute this rating.

Moisture: The moisture is the actual percentage moisture of the grain atharvest.

New Breeding Techniques: New breeding techniques are said of various newtechnologies developed and/or used to create new characteristics inplants through genetic variation, the aim being targeted mutagenesis,targeted introduction of new genes or gene silencing (RdDM). Example ofsuch new breeding techniques are targeted sequence changes facilitatedthru the use of Zinc finger nuclease (ZFN) technology (ZFN-1, ZFN-2 andZFN-3, see U.S. Pat. No. 9,145,565, incorporated by reference in itsentirety), Oligonucleotide directed mutagenesis (ODM), Cisgenesis andintragenesis, RNA-dependent DNA methylation (RdDM, which does notnecessarily change nucleotide sequence but can change the biologicalactivity of the sequence), Grafting (on GM rootstock), Reverse breeding,Agro-infiltration (agro-infiltration “sensu stricto”, agro-inoculation,floral dip), genome editing with endonucleases such as chemicalnucleases, meganucleases, ZFNs, and Transcription Activator-LikeEffector Nucleases (TALENs, see U.S. Pat. Nos. 8,586,363 and 9,181,535,incorporated by reference in their entireties), the CRISPR/Cas system(using such as Cas9, Cas12a/Cpf1, Cas13/C2c2, CasX and CasY; see U.S.Pat. Nos. 8,697,359; 8,771,945; 8,795,965; 8,865,406; 8,871,445;8,889,356; 8,895,308; 8,906,616; 8,932,814; 8,945,839; 8,993,233; and8,999,641, which are all hereby incorporated by reference), engineeredmeganuclease, engineered homing endonucleases, DNA guided genome editing(Gao et al., Nature Biotechnology (2016), doi: 10.1038/nbt.3547,incorporated by reference in its entirety), and Synthetic genomics. Amajor part of today's targeted genome editing, another designation forNew Breeding Techniques, is the applications to induce a DNA doublestrand break (DSB) at a selected location in the genome where themodification is intended. Directed repair of the DSB allows for targetedgenome editing. Such applications can be utilized to generate mutations(e.g., targeted mutations or precise native gene editing) as well asprecise insertion of genes (e.g., cisgenes, intragenes, or transgenes).The applications leading to mutations are often identified assite-directed nuclease (SDN) technology, such as SDN1, SDN2 and SDN3.For SDN1, the outcome is a targeted, non-specific genetic deletionmutation: the position of the DNA DSB is precisely selected, but the DNArepair by the host cell is random and results in small nucleotidedeletions, additions or substitutions. For SDN2, a SDN is used togenerate a targeted DSB and a DNA repair template (a short DNA sequenceidentical to the targeted DSB DNA sequence except for one or a fewnucleotide changes) is used to repair the DSB: this results in atargeted and predetermined point mutation in the desired gene ofinterest. As to the SDN3, the SDN is used along with a DNA repairtemplate that contains new DNA sequence (e.g. gene). The outcome of thetechnology would be the integration of that DNA sequence into the plantgenome. The most likely application illustrating the use of SDN3 wouldbe the insertion of cisgenic, intragenic, or transgenic expressioncassettes at a selected genome location. A complete description of eachof these techniques can be found in the report made by the JointResearch Center (JRC) Institute for Prospective Technological Studies ofthe European Commission in 2011 and titled “New plant breedingtechniques—State-of-the-art and prospects for commercial development”,which is incorporated by reference in its entirety.

Plant cell: Plant cell, as used herein includes plant cells whetherisolated, in tissue culture, or incorporated in a plant or plant part.

Plant habit: This is a visual assessment assigned during the latevegetative to early reproductive stages to characterize the plant's leafhabit. It ranges from decumbent with leaves growing horizontally fromthe stalk to a very upright leaf habit, with leaves growing nearvertically from the stalk.

Plant height: This is a measure of the height of the hybrid from theground to the tip of the tassel, and is measured in centimeters.

Plant intactness: This is a visual assessment assigned to a hybrid orinbred at or close to harvest to indicate the degree that the plant hassuffered disintegration through the growing season. Plants are ratedfrom 1 (poorest) to 9 (best) with poorer scores given for plants thathave more of their leaf blades missing.

Plant part: As used herein, the term “plant part” includes any part ofthe plant including but not limited to leaves, stems, roots, seeds,grains, embryos, pollens, ovules, flowers, ears, cobs, husks, stalks,root tips, anthers, silk, tissue, cells and the like.

Pollen shed: This is a visual rating assigned at flowering to describethe abundance of pollen produced by the anthers. Inbreds are rated 1(poorest) to 9 (best) with the best scores for inbreds with tassels thatshed more pollen during anthesis.

Post-anthesis root lodging: This is a percentage plants that root lodgeafter anthesis: plants that lean from the vertical axis at anapproximately 30° angle or greater.

Pre-anthesis brittle snapping: This is a percentage of “snapped” plantsfollowing severe winds prior to anthesis.

Pre-anthesis root lodging: This is a percentage plants that root lodgeprior to anthesis: plants that lean from the vertical axis at anapproximately 30° angle or greater.

Predicted RAI: This trait for a hybrid, predicted relative maturity(RM), is based on the harvest moisture of the grain. The relativematurity rating is based on a known set of checks and utilizesconventional maturity such as the Comparative Relative Maturity RatingSystem or its similar, the Minnesota Relative Maturity Rating System.

Quantitative trait loci (QTL): Quantitative trait loci refer to geneticloci that control to some degree numerically representable traits thatare usually continuously distributed.

RBDHV, CCR1, CRW2-1: RBDHV, CCR1 or CRW2-1 refers to MON88017, alsoknown as MON88017CCR1, is the transgenic event that, when expressed inmaize, allows the use of glyphosate as a weed control agent. Inaddition, this event produces an endotoxin that is efficacious againstthe corn root worm, Diabrotica virgifera, and certain other Coleopteranlarvae.

Regeneration: Regeneration refers to the development of a plant fromtissue culture.

RMQKZ, RMQKC, RMQKZ, RMQKC, or RMQKD refers to a combination of MON88017(see below) and MON89034 transgenes for insect resistance and gyphosatetolerance. MON89034 is a transgenic event expressed in maize, thatproduces an endotoxin that is efficacious against the European cornborer, Ostrinia nubilalis and certain other Lepidopteran larvae.

RHTTZ, RR2: RHTZZ and RR2 refers to MON603, also known as MON603RR2,better known as NK603, is the designation for the transgenic event that,when expressed in maize, allows the use of glyphosate as a weed controlagent on the crop. Another transgenic event, GA21, when expressed inmaize, also allows the use of glyphosate as a weed control agent on thecrop.

Root lodging: The root lodging is the percentage of plants that rootlodge; i.e., those that lean from the vertical axis at an approximate30° angle or greater would be counted as root lodged.

Seed quality: This is a visual rating assigned to the kernels of theinbred. Kernels are rated 1 (poorest) to 9 (best) with poorer scoresgiven for kernels that are very soft and shriveled with splitting of thepericarp visible and better scores for fully formed kernels.

Seedling vigor: This is the vegetative growth after emergence at theseedling stage, approximately five leaves.

Silking ability: This is a visual assessment given during flowering.Plants are rated on the amount and timing of silk production. Plants arerated from 1 (poorest) to 9 (best) with poorer scores given for plantsthat produce very little silks that are delayed past pollen shed.

Single locus converted (conversion): Single locus converted (conversion)plants refers to plants which are developed by a plant breedingtechnique called backcrossing wherein essentially all of the desiredmorphological and physiological characteristics of a variety arerecovered in addition to the single locus transferred into the varietyvia the backcrossing technique or via genetic engineering. A singlelocus converted plant can also be referred to a plant obtained thoughmutagenesis taught in the present disclosure or through the use of somenew breeding techniques. In some embidments, the single locus convertedplant has essentially all of the desired morphological and physiologicalcharacteristics of the original variety in addition to a single locusconverted by spontaneous and/or artificially induced mutations, which isintroduced and/or transferred into the plant by the plant breedingtechniques such as backcrossing. In other embodiments, the single locusconverted plant has essentially all of the desired morphological andphysiological characteristics of the original variety in addition to asingle locus, gene or nucleotide sequence(s) converted, mutated,modified or engineered through the New Breeding Techniques taughtherein. In the present disclosure, single locus converted (conversion)can be interchangeably referred to single gene converted (conversion).

Stalk lodging: This is the percentage of plants that stalk lodge, i.e.,stalk breakage, as measured by either natural lodging or pushing thestalks and determining the percentage of plants that break off below theear. This is a relative rating of a hybrid to other hybrids forstandability.

Standability: A term referring to the how well a plant remains uprighttowards the end of the growing season. Plants with excessive stalkbreakage and/or root lodging would be considered to have poorstandability.

Stay green: Stay green is the measure of plant health near the time ofblack layer formation (physiological maturity). A high score indicatesbetter late-season plant health.

Transgene: Transgene refers to any nucleotide sequence used in thetransformation of a plant (e.g., maize), animal, or other organism.Thus, a transgene can be a coding sequence, a non-coding sequence, acDNA, a gene or fragment or portion thereof, a genomic sequence, aregulatory element and the like.

Transgenic: A transgenic organism, such as a transgenic plant, is anorganism into which a transgene has been delivered or introduced and thetransgene can be expressed in the transgenic organism to produce aproduct, the presence of which can impart an effect and/or a phenotypein the organism. Where an inbred line has been converted to contain oneor more transgenes by single locus conversion or by directtransformation.

Variety and Cultivar: Variety and Cultivar, which can be interchangeablyused in the present disclosure, refer to a group of similar plants thatby structural or genetic features and/or performance can bedistinguished from other varieties within the same species. A plantvariety as used by one skilled in the art of plant breeding means aplant grouping within a single botanical taxon of the lowest known rankwhich can be defined by the expression of the characteristics resultingfrom a given genotype or combination of phenotypes, distinguished fromany other plant grouping by the expression of at least one of the saidcharacteristics and considered as a unit with regard to its suitabilityfor being propagated unchanged (International Convention for theProtection of New Varieties of Plants).

Yield (Bushels/Acre): The yield is the actual yield of the grain atharvest adjusted to 15.5% moisture.

ZKDDZ: ZKDDZ refers to MON810, also known as MON810Bt or BT1, is thedesignation given by the Monsanto Company (St. Louis, MO) for thetransgenic event that, when expressed in maize, produces an endotoxinthat is efficacious against the European corn borer, Ostrinia nubilalisand certain other Lepidopteran larvae.

DETAILED DESCRIPTION OF THE DISCLOSURE

The disclosures, including the claims, figures and/or drawings, of eachand every patent, patent application, and publication cited herein arehereby incorporated herein by reference in their entireties.

The following description includes information that may be useful inunderstanding the present disclosure. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed disclosures, or that any publication specifically orimplicitly referenced is prior art.

There are numerous steps in the development of any novel, desirableplant germplasm. Plant breeding begins with the analysis and definitionof problems and weaknesses of the current germplasm, the establishmentof program goals, and the definition of specific breeding objectives.The next step is selection of germplasm that possess the traits to meetthe program goals. The goal is to combine in a single variety or hybridan improved combination of desirable traits from the parental germplasm.These important traits may include higher yield, resistance to diseasesand insects, better stalks and roots, tolerance to drought and heat,reduction of grain moisture at harvest as well as better agronomicquality. With mechanical harvesting of many crops, uniformity of plantcharacteristics such as germination and stand establishment, growthrate, maturity and plant and ear height is important.

Choice of breeding or selection methods depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of cultivar used commercially (e.g., F₁ hybrid cultivar, purelinecultivar, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location will be effective,whereas for traits with low heritability, selection should be based onmean values obtained from replicated evaluations of families of relatedplants. Popular selection methods commonly include pedigree selection,modified pedigree selection, mass selection, recurrent selection, andbackcross breeding.

The complexity of inheritance influences choice of breeding method.Backcross breeding is used to transfer one or a few favorable genes fora heritable trait into a desirable cultivar. This approach has been usedextensively for breeding disease-resistant cultivars; nevertheless, itis also suitable for the adjustment and selection of morphologicalcharacters, color characteristics and simply inherited quantitativecharacters such as earliness, plant height or seed size and shape.Various recurrent selection techniques are used to improvequantitatively inherited traits controlled by numerous genes. The use ofrecurrent selection in self-pollinating crops depends on the ease ofpollination, the frequency of successful hybrids from each pollination,and the number of hybrid offspring from each successful cross.

Each breeding program should include a periodic, objective evaluation ofthe efficiency of the breeding procedure. Evaluation criteria varydepending on the goal and objectives, but should include gain fromselection per year based on comparisons to an appropriate standard,overall value of the advanced breeding lines, and number of successfulcultivars produced per unit of input (e.g., per year, per dollarexpended, etc.).

Promising advanced breeding lines are thoroughly tested per se and inhybrid combination and compared to appropriate standards in environmentsrepresentative of the commercial target area(s) for three or more years.The best lines are candidates for use as parents in new commercialcultivars; those still deficient in a few traits may be used as parentsto produce new populations for further selection.

These processes, which lead to the final step of marketing anddistribution, usually take from eight to twelve years from the time thefirst cross is made. Therefore, development of new cultivars is atime-consuming process that requires precise forward planning, efficientuse of resources, and a focus on clear objectives.

A most difficult task is the identification of individuals that aregenetically superior, because for most traits the true genotypic valueis masked by other confounding plant traits or environmental factors.One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to a widely grown standardcultivar. If a single observation is inconclusive, replicatedobservations provide a better estimate of its genetic worth.

The goal of corn breeding is to develop new, unique and superior corninbred lines and hybrids. The breeder initially selects and crosses twoor more parental lines, followed by repeated self pollination or selfingand selection, producing many new genetic combinations. Another methodused to develop new, unique and superior corn inbred lines and hybridsoccurs when the breeder selects and crosses two or more parental lines,followed by haploid induction and chromosome doubling that results inthe development of dihaploid inbred lines. The breeder can theoreticallygenerate billions of different genetic combinations via crossing,selfing and mutations and the same is true for the utilization of thedihaploid breeding method.

Each year, the plant breeder selects the germplasm to advance to thenext generation. This germplasm is grown under unique and differentgeographical, climatic and soil conditions, and further selections arethen made, during and at the end of the growing season. The inbred lineswhich are developed are unpredictable. This unpredictability is becausethe breeder's selection occurs in unique environments, with no controlat the DNA level (using conventional breeding procedures or dihaploidbreeding procedures), and with millions of different possible geneticcombinations being generated. A breeder of ordinary skill in the artcannot predict the final resulting lines he develops, except possibly ina very gross and general fashion. This unpredictability results in theexpenditure of large research funds to develop several superior new corninbred lines.

The development of commercial corn hybrids requires the development ofhomozygous inbred lines, the crossing of these lines, and the evaluationof the crosses. Pedigree breeding and recurrent selection breedingmethods are used to develop inbred lines from breeding populations.Breeding programs combine desirable traits from two or more inbred linesor various broad-based sources into breeding pools from which inbredlines are developed by selfing and selection of desired phenotypes orthrough the dihaploid breeding method followed by the selection ofdesired phenotypes. The new inbreds are crossed with other inbred linesand the hybrids from these crosses are evaluated to determine which havecommercial potential.

Pedigree breeding is used commonly for the improvement ofself-pollinating crops or inbred lines of cross-pollinating crops. Twoparents which possess favorable, complementary traits are crossed toproduce an F₁. An F₂ population is produced by selfing one or severalF₁s or by intercrossing two F₁s (sib mating). Selection of the bestindividuals is usually begun in the F₂ population; then, beginning inthe F₃, the best individuals in the best families are selected.Replicated testing of families, or hybrid combinations involvingindividuals of these families, often follows in the F₄ generation toimprove the effectiveness of selection for traits with low heritability.At an advanced stage of inbreeding (i.e., F₆ and F₇), the best lines ormixtures of phenotypically similar lines are tested for potentialrelease as new cultivars. Similarly, the development of new inbred linesthrough the dihaploid system requires the selection of the best inbredsfollowed by four to five years of testing in hybrid combinations inreplicated plots.

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified or createdby intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued.

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable cultivar or inbredline which is the recurrent parent. The source of the trait to betransferred is called the donor parent. The resulting plant is expectedto have the attributes of the recurrent parent (e.g., cultivar) and thedesirable trait transferred from the donor parent. After the initialcross, individuals possessing the phenotype of the donor parent areselected and repeatedly crossed (backcrossed) to the recurrent parent.The resulting plant is expected to have the attributes of the recurrentparent (e.g., cultivar) and the desirable trait transferred from thedonor parent.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., R. W. Allard, 1960, Principles of Plant Breeding, JohnWiley and Son; Briggs, F. N. and Knowles, P. F. 1967. Introduction toPlant Breeding. Reinhold Publishing Corporation; N. W. Simmonds, 1979,Principles of Crop Improvement, Longman Group Limited; W. R. Fehr, 1987,Principles of Crop Development, Macmillan Publishing Co.; N. F. Jensen,1988, Plant Breeding Methodology, John Wiley & Sons).

Proper testing should detect any major faults and establish the level ofsuperiority or improvement over current cultivars. In addition toshowing superior performance, there must be a demand for a new cultivarthat is compatible with industry standards or which creates a newmarket. The introduction of a new cultivar will incur additional coststo the seed producer, the grower, processor and consumer for specialadvertising and marketing, altered seed and commercial productionpractices, and new product utilization. The testing preceding release ofa new cultivar should take into consideration research and developmentcosts as well as technical superiority of the final cultivar. Forseed-propagated cultivars, it must be feasible to produce seed easilyand economically.

Once the inbreds that give the best hybrid performance have beenidentified, the hybrid seed can be reproduced indefinitely as long asthe homogeneity of the inbred parent is maintained. A single-crosshybrid is produced when two inbred lines are crossed to produce the F₁progeny. A double-cross hybrid is produced from four inbred linescrossed in pairs (A×B and C×D) and then the two F₁ hybrids are crossedagain (A×B)×(C×D). Much of the hybrid vigor exhibited by F₁ hybrids islost in the next generation (F₂). Consequently, seed from hybridvarieties is not used for planting stock.

Hybrid corn seed is typically produced by a male sterility system or byincorporating manual or mechanical detasseling. Alternate strips of twocorn inbreds are planted in a field, and the pollen-bearing tassels areremoved from one of the inbreds (female). Providing that there issufficient isolation from sources of foreign corn pollen, the ears ofthe detasseled inbred will be fertilized only from the other inbred(male), and the resulting seed is therefore hybrid and will form hybridplants.

The laborious, and occasionally unreliable, detasseling process can beavoided by using cytoplasmic male-sterile (CMS) inbreds. Plants of a CMSinbred are male sterile as a result of factors resulting from thecytoplasmic, as opposed to the nuclear, genome. Thus, thischaracteristic is inherited exclusively through the female parent incorn plants, since only the female provides cytoplasm to the fertilizedseed. CMS plants are fertilized with pollen from another inbred that isnot male-sterile. Pollen from the second inbred may or may notcontribute genes that make the hybrid plants male-fertile. Seed fromdetasseled fertile corn and CMS produced seed of the same hybrid can beblended to insure that adequate pollen loads are available forfertilization when the hybrid plants are grown.

There are several methods of conferring genetic male sterilityavailable, such as multiple mutant genes at separate locations withinthe genome that confer male sterility, as disclosed in U.S. Pat. Nos.4,654,465 and 4,727,219 to Brar et al. These and all patents referred toare incorporated by reference. In addition to these methods, Albertsenet al., U.S. Pat. No. 5,432,068, has developed a system of nuclear malesterility which includes: identifying a gene which is critical to malefertility, silencing this native gene which is critical to malefertility; removing the native promoter from the essential malefertility gene and replacing it with an inducible promoter; insertingthis genetically engineered gene back into the plant; and thus creatinga plant that is male sterile because the inducible promoter is not “on”resulting in the male fertility gene not being transcribed. Fertility isrestored by inducing, or turning “on,” the promoter, which in turnallows the gene that confers male fertility to be transcribed.

There are many other methods of conferring genetic male sterility in theart, each with its own benefits and drawbacks. These methods use avariety of approaches such as delivering into the plant a gene encodinga cytotoxic substance associated with a male tissue specific promoter oran anti-sense system in which a gene critical to fertility is identifiedand an antisense to that gene is inserted in the plant (see,Fabinjanski, et al. EPO 89/0301053.8 publication number 329,308 and PCTapplication PCT/CA90/00037 published as WO 90/08828).

Another version useful in controlling male sterility makes use ofgametocides. Gametocides are not a genetic system, but rather a topicalapplication of chemicals. These chemicals affect cells that are criticalto male fertility. The application of these chemicals affects fertilityin the plants only for the growing season in which the gametocide isapplied (see Carlson, G. R., U.S. Pat. No. 4,936,904). Application ofthe gametocide, timing of the application, and genotype often limit theusefulness of the approach.

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification.

Inbred Corn Line KW4ER1502

Inbred corn line KW4ER1502 is a corn inbred with superiorcharacteristics, and provides very good parental lines in crosses forproducing first generation (F1) hybrid corn. Inbred corn line KW4ER1502is best adapted to the East, Central, and Western regions of the UnitedStates Corn Belt in the zones that are commonly referred to as Zones 3,4 and 5. Hybrids that are adapted to these maturity zones can be grownon a significant number of acres as it relates to the total of the USAcorn acres.

KW4ER1502 is an inbred corn line with high yield potential in hybrids.Hybrids with inbred corn line KW4ER1502 as one parental line produceuniform, consistent sized ears. Often these hybrid combinations resultin plants which are appreciably better than average for stalk and rootstrength, and grain yield when compared to inbred lines of similarmaturity and geographical adaptability. Some of the criteria used toselect ears in various generations include: yield, yield to harvestmoisture ratio, stalk quality, root quality, disease tolerance withemphasis on grey leaf spot, test weight, late season plant greenness,late season plant intactness, ear retention, ear height, pollen sheddingability, silking ability, and corn borer tolerance. During thedevelopment and selection of the line, crosses were made to inbredtesters for the purpose of estimating the line's general and specificcombining ability, and evaluations were run by the Morris, MinnesotaStation. The inbred was evaluated further as a line and in numerouscrosses by the Morris station and other research stations across theCorn Belt. The inbred has proven to have an excellent combining abilityin hybrid combinations.

Inbred corn line KW4ER1502 has shown uniformity and stability for thetraits, within the limits of environmental influence for the traits.These lines have been increased with continued observation foruniformity of plant type. Inbred corn line KW4ER1502 has the followingmorphologic and other characteristics (based primarily on data collectedat Morris, Minnesota unless otherwise specified).

TABLE 1 Description Information on Inbred Corn Line KW4ER1502 varietyGeneral 1 Type: KW4ER1502 is a yellow, dent corn inbred Plant 2. Regionwhere developed: Alexandria, Romania Information 3. Maturity: 98 RM HeatUnits From planting to 50% of plants in silk: 1471 GDD From planting to50% of plants in pollen: 1460 GDD Plant 1. Plant height to tassel tip:218.9 cm 2. Ear height to base of top ear: 80.9 cm 3. Average length oftop ear internode: 12.6 cm 4. Average number of tillers: 0 5 Averagenumber of ears per stalk: 1.5 6. Anthocyanin of brace roots: FaintLeaf 1. Width of ear node leaf: 8.9 cm 2. Length of ear node leaf: 79.1cm 3. Number of leaves above top ear: 5.8 4. Leaf angle (from 2nd leafabove ear at anthesis to stalk above leaf): 20.2° 5. Leaf color: Munsell5GY 4/6 6. Leaf sheath pubescence (Rated on scale from 1 = none to 9 =like peach fuzz): 8 7. Marginal waves (Rated on scale from 1 = none to 9= many): 4 8. Longitudinal creases (Rated on scale from 1 = none to 9 =many): 7 Tassel 1 Number of lateral branches: 8.3 2. Branch angle fromcentral spike: 20.80 3. Tassel length (from top leaf collar to tasseltop): 39.4 cm Pollen shed (Rated on scale from 0 = male sterile to 9 =heavy shed): 7 5 Anther color: Munsell 5R 5/2 6. Glume color: Munsell5GY 5/10 7. Tassel glume bands color: Present Ear 1. Silk color (3 daysafter emergence): Munsell 2.5GY 8/10 (Unhusked 2. Fresh husk color (25days after 50% silking): Munsell 5GY 7/6 Data) 3. Dry husk color (65days after 50% silking): Munsell 2.5Y 8/6 4. Position of ear: Upright 5.Husk tightness (Rated on scale from 1 = very loose to 9 = very tight): 86. Husk extension at harvest: Short(<8 cm) Ear 1. Ear length: 15.4 cm(Husked 2. Ear diameter at mid-point: 41.2 mm Ear Data) 3 Ear weight:120.1 g 4. Number of kernel rows: 14 5. Row alignment: Straight 6. Eartaper: Average Kernel 1. Kernel length: 12.2 mm (Dried) 2. Kernel width:8.3 mm 3. Kernel thickness: 5.6 mm 4 Hard endosperm color: Munsell 2.5Y7/10 5. Endosperm type: Normal 6. Weight per 100 kernels (unsizedsample): 34.7 g Cob 1. Cob diameter at mid-point: 23.7 mm 2. Cob color:Munsell 10R 5/8 Agronomic 1. Dropped ears (at 65 days after anthesis):0% Traits 2. Pre-anthesis brittle snapping: 0% 3 Pre-anthesis rootlodging: 0% 4. Post-anthesis root lodging (at 65 days after harvest): 0%

Further Embodiments of the Disclosure

This disclosure is also directed to methods for producing a corn plantby crossing a first parent corn plant with a second parent corn plantwherein either the first or second parent corn plant is an inbred cornplant of inbred corn line KW4ER1502. Further, both first and secondparent corn plants can come from the inbred corn line KW4ER1502. Whenself-pollinated, or crossed with another inbred corn line KW4ER1502plant, inbred corn line KW4ER1502 will be stable while when crossed withanother, different corn line, an F₁ hybrid seed is produced. Suchmethods of hybridization and self-pollination of corn are well known tothose skilled in the art of corn breeding.

An inbred corn line has been produced through several cycles ofself-pollination and is therefore to be considered as a homozygous line.An inbred line can also be produced though the dihaploid system whichinvolves doubling the chromosomes from a haploid plant thus resulting inan inbred line that is genetically stable (homozygous) and can bereproduced without altering the inbred line. A hybrid variety isclassically created through the fertilization of an ovule from an inbredparental line by the pollen of another, different inbred parental line.Due to the homozygous state of the inbred line, the produced gametescarry a copy of each parental chromosome. As both the ovule and thepollen bring a copy of the arrangement and organization of the genespresent in the parental lines, the genome of each parental line ispresent in the resulting F₁ hybrid, theoretically in the arrangement andorganization created by the plant breeder in the original parental line.

As long as the homozygosity of the parental lines is maintained, theresulting hybrid cross is stable. The F₁ hybrid is then a combination ofphenotypic characteristics issued from two arrangement and organizationof genes, both created by one skilled in the art through the breedingprocess.

Still further, this disclosure also is directed to methods for producingan inbred corn line KW4ER1502-derived corn plant by crossing inbred cornline KW4ER1502 with a second corn plant and growing the progeny seed,and repeating the crossing and growing steps with the inbred corn lineKW4ER1502-derived plant from 0 to 7 times. Thus, any such methods usingthe inbred corn line KW4ER1502 are part of this disclosure: selfing,backcrosses, hybrid production, crosses to populations, and the like.All plants produced using inbred corn line KW4ER1502 as a parent arewithin the scope of this disclosure, including plants derived frominbred corn line KW4ER1502. Advantageously, the inbred corn line is usedin crosses with other, different, corn inbreds to produce firstgeneration (F₁) corn hybrid seeds and plants with superiorcharacteristics.

It should be understood that the inbred can, through routinemanipulation of cytoplasmic or other factors, be produced in amale-sterile form. Such embodiments are also contemplated within thescope of the present claims. As used herein, the term plant includesplant cells, plant protoplasts, plant cell tissue cultures from whichcorn plants can be regenerated, plant calli, plant clumps and plantcells that are intact in plants or parts of plants, such as embryos,pollen, ovules, flowers, kernels, seeds, ears, cobs, leaves, husks,stalks, roots, root tips, anthers, silk and the like.

Duncan, et al., (Planta, 1985, 165:322-332) indicates that 97% of theplants cultured that produced callus were capable of plant regeneration.Subsequent experiments with both inbreds and hybrids produced 91%regenerable callus that produced plants. In a further study in 1988,Songstad, et al. (Plant Cell Reports, 7:262-265) reports several mediaadditions that enhance regenerability of callus of two inbred lines.Other published reports also indicated that “nontraditional” tissues arecapable of producing somatic embryogenesis and plant regeneration. K. V.Rao et al., (Maize Genetics Cooperation Newsletter, 1986, 60:64-65)refer to somatic embryogenesis from glume callus cultures and B. V.Conger, et al. (Plant Cell Reports, 1987, 6:345-347) indicate somaticembryogenesis from the tissue cultures of corn leaf segments. Thus, itis clear from the literature that the state of the art is such thatthese methods of obtaining plants are routinely used and have a veryhigh rate of success.

Tissue culture of corn is also described in European Patent Application,publication 160,390 and in Green and Rhodes, Maize for BiologicalResearch, Plant Molecular Biology Association, Charlottesville, V A,1982, 367-372. Thus, another aspect of this disclosure is to providecells which upon growth and differentiation produce corn plants havingthe physiological and morphological characteristics of inbred corn lineKW4ER1502.

The utility of inbred corn line KW4ER1502 also extends to crosses withother species. Commonly, suitable species will be of the familyGraminaceae, and especially of the genera Zea, Tripsacum, Coix,Schlerachne, Polytoca, Chionachne, and Trilobachne, of the tribeMaydeae. Potentially suitable for crosses with KW4ER1502 may be thevarious varieties of grain Sorghum, Sorghum bicolor (L.) Moench.

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and expressforeign genes, or additional, or modified versions of native, orendogenous, genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Such foreignadditional and/or modified genes are referred to herein collectively as“transgenes.” Over the last fifteen to twenty years several methods forproducing transgenic plants have been developed, and the presentdisclosure, in particular embodiments, also relates to transformedversions of the claimed inbred line. An embodiment of the presentdisclosure comprises at least one transformation event in inbred cornline KW4ER1502.

Plant transformation involves the construction of an expression vectorwhich will function in plant cells. Such a vector comprises DNAcomprising a gene under control of, or operatively linked to, aregulatory element (for example, a promoter). The expression vector maycontain one or more such operably linked gene/regulatory elementcombinations. The vector(s) may be in the form of a plasmid, and can beused alone or in combination with other plasmids, to provide transformedcorn plants, using transformation methods as described below toincorporate transgenes into the genetic material of the corn plant(s).

Expression Vectors for Corn Transformation: Marker Genes

Expression vectors include at least one genetic marker, operably linkedto a regulatory element (a promoter, for example) that allowstransformed cells containing the marker to be either recovered bynegative selection, i.e., inhibiting growth of cells that do not containthe selectable marker gene, or by positive selection, i.e., screeningfor the product encoded by the genetic marker. Many commonly usedselectable marker genes for plant transformation are well known in thetransformation arts, and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or a herbicide, or genes that encode an altered targetwhich is insensitive to the inhibitor. A few positive selection methodsare also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII) gene, isolated from transposonTn5, which, when placed under the control of plant regulatory signals,confers resistance to kanamycin (Fraley et al., Proc. Natl. Acad. Sci.U.S.A., 80:4803 (1983)). Another commonly used selectable marker gene isthe hygromycin phosphotransferase gene which confers resistance to theantibiotic hygromycin (Vanden Elzen et al., Plant Mol. Biol., 5:299(1985)).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, and aminoglycoside-3′-adenyltransferase, the bleomycin resistance determinant (Hayford et al., PlantPhysiol. 86:1216 (1988), Jones et al., Mol. Gen. Genet., 210:86 (1987),Svab et al., Plant Mol. Biol. 14:197 (1990), and Hille et al., PlantMol. Biol. 7:171 (1986)). Other selectable marker genes conferresistance to herbicides such as glyphosate, glufosinate or bromoxynil(Comai et al., Nature 317:741-744 (1985), Gordon-Kamm et al., Plant Cell2:603-618 (1990) and Stalker et al., Science 242:419-423 (1988)).

Selectable marker genes for plant transformation that are not ofbacterial origin include, for example, mouse dihydrofolate reductase,plant 5-enolpyruvylshikimate-3-phosphate synthase and plant acetolactatesynthase (Eichholtz et al., Somatic Cell Mol. Genet. 13:67 (1987), Shahet al., Science 233:478 (1986), and Charest et al., Plant Cell Rep.8:643 (1990)).

Another class of marker genes for plant transformation requiresscreening of presumptively transformed plant cells rather than directgenetic selection of transformed cells for resistance to a toxicsubstance such as an antibiotic. These genes are particularly useful toquantify or visualize the spatial pattern of expression of a gene inspecific tissues and are frequently referred to as reporter genesbecause they can be fused to a gene or gene regulatory sequence for theinvestigation of gene expression. Commonly used genes for screeningpresumptively transformed cells include beta-glucuronidase (GUS),beta-galactosidase, luciferase, and chloramphenicol acetyltransferase(Jefferson, R. A., Plant Mol. Biol. Rep. 5:387 (1987), Teeri et al.,EMBO J. 8:343 (1989), Koncz et al., Proc. Natl. Acad. Sci U.S.A. 84:131(1987), and DeBlock et al., EMBO J. 3:1681 (1984). Another approach tothe identification of relatively rare transformation events has been useof a gene that encodes a dominant constitutive regulator of the Zea maysanthocyanin pigmentation pathway (Ludwig et al., Science 247:449(1990)).

In vivo methods for visualizing GUS activity that do not requiredestruction of plant tissue are also available. However, these in vivomethods for visualizing GUS activity have not proven useful for recoveryof transformed cells because of low sensitivity, high fluorescentbackgrounds and limitations associated with the use of luciferase genesas selectable markers.

A gene encoding Green Fluorescent Protein (GFP) has been utilized as amarker for gene expression in prokaryotic and eukaryotic cells (Chalfieet al., Science 263:802 (1994)). GFP and mutants of GFP may be used asscreenable markers.

Expression Vectors for Corn Transformation: Promoters

Genes included in expression vectors must be driven by nucleotidesequence comprising a regulatory element, for example, a promoter.Several types of promoters are now well known in the transformationarts, as are other regulatory elements that can be used alone or incombination with promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain organs,such as leaves, roots, seeds and tissues such as fibers, xylem vessels,tracheids, or sclerenchyma. Such promoters are referred to as“tissue-preferred.” Promoters which initiate transcription only incertain tissue are referred to as “tissue-specific.” A “cell-type”specific promoter primarily drives expression in certain cell types inone or more organs, for example, vascular cells in roots or leaves. An“inducible” promoter is a promoter which is under environmental control.Examples of environmental conditions that may affect transcription byinducible promoters include anaerobic conditions or the presence oflight. Tissue-specific, tissue-preferred, cell-type specific, andinducible promoters constitute the class of “non-constitutive”promoters. A “constitutive” promoter is a promoter which is active undermost environmental conditions.

A. Inducible Promoters—An inducible promoter is operably linked to agene for expression in corn. Optionally, the inducible promoter isoperably linked to a nucleotide sequence encoding a signal sequencewhich is operably linked to a gene for expression in corn. With aninducible promoter the rate of transcription increases in response to aninducing agent. Any inducible promoter can be used in the instantdisclosure. See Ward et al., Plant Mol. Biol. 22:361-366 (1993).Exemplary inducible promoters include, but are not limited to, that fromthe ACEI system which responds to copper (Mett et al., Proc. Natl. Acad.Sci. U.S.A. 90:4567-4571 (1993)); In2 gene from maize which responds tobenzenesulfonamide herbicide safeners (Gatz et al., Mol. Gen. Genetics243:32-38 (1994)) or Tet repressor from Tn10 (Gatz et al., Mol. Gen.Genetics 227:229-237 (1991)). A particularly preferred induciblepromoter is a promoter that responds to an inducing agent to whichplants do not normally respond. An exemplary inducible promoter is theinducible promoter from a steroid hormone gene, the transcriptionalactivity of which is induced by a glucocorticosteroid hormone (Schena etal., Proc. Natl. Acad. Sci. U.S.A. 88:0421 (1991)).

B. Constitutive Promoters—A constitutive promoter is operably linked toa gene for expression in corn or the constitutive promoter is operablylinked to a nucleotide sequence encoding a signal sequence which isoperably linked to a gene for expression in corn. Many differentconstitutive promoters can be utilized in the instant disclosure.Exemplary constitutive promoters include, but are not limited to, thepromoters from plant viruses such as the 35S promoter from CaMV (Odellet al., Nature 313:810-812 (1985)) and the promoters from such genes asrice actin (McElroy et al., Plant Cell 2:163-171 (1990)); ubiquitin(Christensen et al., Plant Mol. Biol. 12:619-632 (1989) and Christensenet al., Plant Mol. Biol. 18:675-689 (1992)); pEMU (Last et al., Theor.Appl. Genet. 81:581-588 (1991)); MAS (Velten et al., EMBO J. 3:2723-2730(1984)) and maize H3 histone (Lepetit et al., Mol. Gen. Genetics231:276-285 (1992) and Atanassova et al., Plant Journal 2 (3): 291-300(1992)).

The ALS promoter, Xba1/Ncol fragment 5′ to the Brassica napus ALS3structural gene (or a nucleotide sequence similarity to said Xba1/Ncolfragment), represents a particularly useful constitutive promoter. SeePCT application WO96/30530.

C. Tissue-specific or Tissue-preferred Promoters—A tissue-specificpromoter is operably linked to a gene for expression in corn.Optionally, the tissue-specific promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in corn. Plants transformed with a gene ofinterest operably linked to a tissue-specific promoter produce theprotein product of the transgene exclusively, or preferentially, in aspecific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant disclosure. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferred promoter,such as that from the phaseolin gene (Murai et al., Science 23:476-482(1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. U.S.A.82:3320-3324 (1985)); a leaf-specific and light-induced promoter such asthat from cab or rubisco (Simpson et al., EMBO J. 4(11):2723-2729 (1985)and Timko et al., Nature 318:579-582 (1985)); an anther-specificpromoter such as that from LAT52 (Twell et al., Mol. Gen. Genetics217:240-245 (1989)); a pollen-specific promoter such as that from Zm13or a microspore-preferred promoter such as that from apg (Twell et al.,Sex. Plant Reprod. 6:217-224 (1993)).

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall ormitochondrion or for secretion into the apoplast, is accomplished bymeans of operably linking the nucleotide sequence encoding a signalsequence to the 5′ and/or 3′ region of a gene encoding the protein ofinterest. Targeting sequences at the 5′ and/or 3′ end of the structuralgene may determine, during protein synthesis and processing, where theencoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample Becker et al., Plant Mol. Biol. 20:49 (1992), Knox, C., et al.,Plant Mol. Biol. 9:3-17 (1987), Lerner et al., Plant Physiol. 91:124-129(1989), Fontes et al., Plant Cell 3:483-496 (1991), Matsuoka et al.,Proc. Natl. Acad. Sci. 88:834 (1991), Gould et al., J. Cell. Biol.108:1657 (1989), Creissen et al., Plant J. 2:129 (1991), Kalderon, etal., Cell 39:499-509 (1984), Stiefel, et al., Plant Cell 2:785-793(1990).

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present disclosure, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem. 114:92-6(1981).

According to a preferred embodiment, the transgenic plant provided forcommercial production of foreign protein is corn. In another preferredembodiment, the biomass of interest is seed. For the relatively smallnumber of transgenic plants that show higher levels of expression, agenetic map can be generated, primarily via conventional RFLP, PCR andSSR analysis, which identifies the approximate chromosomal location ofthe integrated DNA molecule. For exemplary methodologies in this regard,see Glick and Thompson, Methods in Plant Molecular Biology andBiotechnology CRC Press, Boca Raton 269:284 (1993). Map informationconcerning chromosomal location is useful for proprietary protection ofa subject transgenic plant. If unauthorized propagation is undertakenand crosses made with other germplasm, the map of the integration regioncan be compared to similar maps for suspect plants, to determine if thelatter have a common parentage with the subject plant. Map comparisonswould involve hybridizations, RFLP, PCR, SSR and sequencing, all ofwhich are conventional techniques.

Likewise, by means of the present disclosure, agronomic genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below:

1. Genes that Confer Resistance to Pests or Disease and that Encode:

A. Plant disease resistance genes. Plant defences are often activated byspecific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant inbred line can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example Jones et al., Science 266:789 (1994)(cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum);Martin et al., Science 262:1432 (1993) (tomato Pto gene for resistanceto Pseudomonas syringae pv. tomato encodes a protein kinase); Mindrinoset al., Cell 78:1089 (1994) (Arabidopsis RSP2 gene for resistance toPseudomonas syringae).

B. A Bacillus thuringiensis protein, a derivative thereof or a syntheticpolypeptide modeled thereon. See, for example, Geiser et al., Gene48:109 (1986), who disclose the cloning and nucleotide sequence of a Btalpha-endotoxin gene. Moreover, DNA molecules encoding alpha-endotoxingenes can be purchased from American Type Culture Collection, Manassas,Virginia, for example, under ATCC Accession Numbers 40098, 67136, 31995and 31998.

C. A lectin. See, for example, the article by Van Damme et al., PlantMolec. Biol. 24:25 (1994), who disclose the nucleotide sequences ofseveral Clivia miniata mannose-binding lectin genes.

D. A vitamin-binding protein such as avidin. See PCT application US93/06487. The application teaches the use of avidin and avidinhomologues as larvicides against insect pests.

E. An enzyme inhibitor, for example, a protease or proteinase inhibitoror an amylase inhibitor. See, for example, Abe et al., J. Biol. Chem.262:16793 (1987) (nucleotide sequence of rice cysteine proteinaseinhibitor), Huub et al., Plant Molec. Biol. 21:985 (1993) (nucleotidesequence of cDNA encoding tobacco proteinase inhibitor I), Sumitani etal., Biosci. Biotech. Biochem. 57:1243 (1993) (nucleotide sequence ofStreptomyces nitrosporeus alpha-amylase inhibitor).

F. An insect-specific hormone or pheromone such as an ecdysteroid orjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

G. An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosures of Pratt et al., Biochem. Biophys. Res. Comm. 163:1243(1989) (an allostatin is identified in Diploptera puntata). See alsoU.S. Pat. No. 5,266,317 to Tomalski et al., who disclose genes encodinginsect-specific, paralytic neurotoxins.

H. An insect-specific venom produced in nature by a snake, a wasp, etc.For example, see Pang et al., Gene 116:165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

I. An enzyme responsible for a hyper-accumulation of a monoterpene, asesquiterpene, a steroid, a hydroxamic acid, a phenylpropanoidderivative or another non-protein molecule with insecticidal activity.

J. An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 in the name of Scott et al., which discloses thenucleotide sequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Numbers 39637 and 67152. See also Kramer et al., InsectBiochem. Molec. Biol. 23:691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hornworm chitinase, and Kawalleck et al.,Plant Molec. Biol. 21:673 (1993), who provide the nucleotide sequence ofthe parsley ubi4-2 polyubiquitin gene.

K. A molecule that stimulates signal transduction. For example, see thedisclosure by Botella et al., Plant Molec. Biol. 24:757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess etal., Plant Physiol. 104:1467 (1994), who provide the nucleotide sequenceof a maize calmodulin cDNA clone.

L. A hydrophobic moment peptide. See PCT application WO95/16776(disclosure of peptide derivatives of Tachyplesin which inhibit fungalplant pathogens) and PCT application WO 95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance).

M. A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure of Jaynes et al., Plant Sci 89:43 (1993), ofheterologous expression of a cecropin-beta, lytic peptide analog torender transgenic tobacco plants resistant to Pseudomonas solanacearum.

N. A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruseS. See Beachy et al., Ann. Rev. Phytopathol.28:451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus and tobacco mosaic virus.

O. An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect.

P. A virus-specific antibody. See, for example, Tavladoraki et al.,Nature 366:469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

Q. A developmental-arrestive protein produced in nature by a pathogen ora parasite. Thus, fungal endo-alpha-1,4-D-polygalacturonases facilitatefungal colonization and plant nutrient release by solubilising plantcell wall homo-alpha-1,4-D-galacturonase. See Lamb et al., BioTechnology10:1436 (1992). The cloning and characterization of a gene which encodesa bean endopolygalacturonase-inhibiting protein is described by Toubartet al., Plant JH. 2:367 (1992).

R. A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann et al., BioTechnology 10:305 (1992), have shown thattransgenic plants expressing the barley ribosome-inactivating gene havean increased resistance to fungal disease.

2. Genes that Confer Resistance to an Herbicide, for Example:

A. An herbicide that inhibits the growing point or meristem, such as animidazolinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee et al.,EMBO J. 7:1241 (1988), and Miki et al., Theor. Appl. Genet. 80:449(1990), respectively.

B. Glyphosate (resistance conferred by mutant5-enolpyruvylshikimate-3-phosphate synthase (EPSP) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase (PAT) and Streptomyceshygroscopicus (PAT bar genes), and pyridinoxy or phenoxy propionic acidsand cyclohexones (ACCase inhibitor-encoding genes). See, for example,U.S. Pat. No. 4,940,835 to Shah et al., which discloses the nucleotidesequence of a form of EPSP which can confer glyphosate resistance. A DNAmolecule encoding a mutant aroA gene can be obtained under ATCCaccession number 39256, and the nucleotide sequence of the mutant geneis disclosed in U.S. Pat. No. 4,769,061 to Comai. European patentapplication No. 0 333 033 to Kumada et al., and U.S. Pat. No. 4,975,374to Goodman et al., disclose nucleotide sequences of glutamine synthetasegenes which confer resistance to herbicides such as L-phosphinothricin.The nucleotide sequence of a PAT gene is provided in Europeanapplication No. 0 242 246 to Leemans et al. DeGreef et al.,BioTechnology 7:61 (1989), describe the production of transgenic plantsthat express chimeric bar genes coding for PAT activity. Exemplary ofgenes conferring resistance to phenoxy propionic acids and cyclohexones,such as sethoxydim and haloxyfop are the ABC5-S1, ABC5-S2 and ABC5-S3genes described by Marshall et al., Theor. Appl. Genet. 83:435 (1992).

C. An herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) or a benzonitrile (nitrilase gene). Przibilla et al.,Plant Cell 3:169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC AccessionNumbers 53435, 67441, and 67442. Cloning and expression of DNA codingfor a glutathione S-transferase is described by Hayes et al., Biochem.J. 285:173 (1992).

3. Genes that Confer or Contribute to a Value-Added Trait, Such as:

A. Modified fatty acid metabolism, for example, by transforming a plantwith an antisense gene of stearyl-ACP desaturase to increase stearicacid content of the plant. See Knutzon et al., Proc. Natl. Acad. Sci.U.S.A. 89:2624 (1992)

B. Increased resistance to high light stress such as photo-oxidativedamages, for example by transforming a plant with a gene coding for aprotein of the Early Light Induced Protein family (ELIP) as described inWO 03/074713 in the name of Biogemma.

C. Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch. See Shiroza et al., J. Bact. 170:810 (1988)(nucleotide sequence of Streptococcus mutants fructosyltransferasegene), Steinmetz et al., Mol. Gen. Genet. 20:220 (1985) (nucleotidesequence of Bacillus subtilis levansucrase gene), Pen et al.,BioTechnology 10:292 (1992) (production of transgenic plants thatexpress Bacillus licheniformis α-amylase), Elliot et al., Plant Molec.Biol. 21:515 (1993) (nucleotide sequences of tomato invertase genes),Søgaard et al., J. Biol. Chem. 268:22480 (1993) (site-directedmutagenesis of barley alpha-amylase gene), and Fisher et al., PlantPhysiol. 102:1045 (1993) (maize endosperm starch branching enzyme II).

D. Increased resistance/tolerance to water stress or drought, forexample, by transforming a plant to create a plant having a modifiedcontent in ABA-Water-Stress-Ripening-Induced proteins (ARS proteins) asdescribed in WO01/83753 in the name of Biogemma, or by transforming aplant with a nucleotide sequence coding for a phosphoenolpyruvatecarboxylase as shown in WO02/081714. The tolerance of corn to droughtcan also be increased by an overexpression of phosphoenolpyruvatecarboxylase (PEPC-C4), obtained, for example from Sorghum.

E. Increased content of cysteine and glutathione, useful in theregulation of sulfur compounds and plant resistance against variousstresses such as drought, heat or cold, by transforming a plant with agene coding for an Adenosine 5′ Phosphosulfate as shown in WO01/49855.

F. Increased nutritional quality, for example, by introducing a zeingene which genetic sequence has been modified so that its proteinsequence has an increase in lysine and proline. The increasednutritional quality can also be attained by introducing into the maizeplant an albumin 2S gene from sunflower that has been modified by theaddition of the KDEL peptide sequence to keep and accumulate the albuminprotein in the endoplasmic reticulum.

G. Decreased phytate content: 1) Introduction of a phytase-encoding genewould enhance breakdown of phytate, adding more free phosphate to thetransformed plant. For example, see Van Hartingsveldt et al., Gene127:87 (1993), for a disclosure of the nucleotide sequence of anAspergillus niger phytase gene. 2) A gene could be introduced thatreduced phytate content. In maize, this, for example, could beaccomplished, by cloning and then reintroducing DNA associated with thesingle allele which is responsible for maize mutants characterized bylow levels of phytic acid. See Raboy et al., Maydica 35:383 (1990).

4. Genes that Control Male Sterility

A. Introduction of a deacetylase gene under the control of atapetum-specific promoter and with the application of the chemicalN-Ac-PPT. See international publication W01/29237.

B. Introduction of various stamen-specific promoters. See internationalpublications WO92/13956 and WO92/13957.

C. Introduction of the barnase and the barstar genes. See Paul et al.,Plant Mol. Biol. 19:611-622, 1992).

Examples of Transgenes

MON810, also known as MON810Bt or BT1, is the designation given by theMonsanto Company (St. Louis, MO) for the transgenic event that, whenexpressed in maize, produces an endotoxin that is efficacious againstthe European corn borer, Ostrinia nubilalis and certain otherLepidopteran larvae.

MON603, also known as MON603RR2, better known as NK603, is thedesignation for the transgenic event that, when expressed in maize,allows the use of glyphosate as a weed control agent on the crop.Another transgenic event, GA21, when expressed in maize, also allows theuse of glyphosate as a weed control agent on the crop.

MON89034, a designation given by the Monsanto Company (St. Louis, MO)for the transgenic event that, when expressed in maize, produces anendotoxin that is efficacious against the European corn borer, Ostrinianubilalis, fall armyworm, Spodoptera frugiperda, and certain otherLepidopteran larvae.

MON88017, also known as MON88017CCR1, is the transgenic event that, whenexpressed in maize, allows the use of glyphosate as a weed controlagent. In addition, this event produces an endotoxin that is efficaciousagainst the corn root worm, Diabrotica virgifera, and certain otherColeopteran larvae.

HERCULEX Corn Borer, better known as HX1 or TC1507, is the designationfor the transgenic event that, when expressed in maize, produces anendotoxin that is efficacious against the European corn borer, Ostrinianubilalis, and certain other Lepidopteran larvae. In addition, thetransgenic event was developed to allow the crop to be tolerant to theuse of glufosinate ammonium, the active ingredient in phosphinothricinherbicides.

HERCULEX Root Worm, or DAS-59122-7, is the designation for thetransgenic event that, when expressed in maize, produces an endotoxinthat is efficacious against the corn root worm, Diabrotica virgifera,and certain other Coleopteran larvae. In addition, the transgenic eventwas developed to allow the crop to be tolerant to the use of glufosinateammonium, the active ingredient in phosphinothricin herbicides.

T25 is the designation for the transgenic event that, when expressed inmaize, allows the crop to be tolerant to the use of glufosinateammonium, the active ingredient in phosphinothricin herbicides.

Methods for Corn Transformation

Numerous methods for plant transformation have been developed, includingbiological and physical plant transformation protocols. See, forexample, Miki et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, GlickB. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages67-88. In addition, expression vectors and in vitro culture methods forplant cell or tissue transformation and regeneration of plants areavailable. See, for example, Gruber et al., “Vectors for PlantTransformation” in Methods in Plant Molecular Biology and Biotechnology,Glick B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993)pages 89-119.

A. Agrobacterium-mediated Transformation—One method for introducing anexpression vector into plants is based on the natural transformationsystem of Agrobacterium. See, for example, Horsch et al., Science227:1229 (1985). A. tumefaciens and A. rhizogenes are plant pathogenicsoil bacteria which genetically transform plant cells. The Ti and Riplasmids of A. tumefaciens and A. rhizogenes, respectively, carry genesresponsible for genetic transformation of the plant. See, for example,Kado, C. I., Crit. Rev. Plant Sci. 10:1 (1991). Descriptions ofAgrobacterium vector systems and methods for Agrobacterium-mediated genetransfer are provided by Gruber et al., supra, Miki et al., supra, andMoloney et al., Plant Cell Reports 8:238 (1989). See also, U.S. Pat. No.5,591,616 issued Jan. 7, 1997.

B. Direct Gene Transfer—Despite the fact the host range forAgrobacterium-mediated transformation is broad, some major cereal cropspecies and gymnosperms have generally been recalcitrant to this mode ofgene transfer, even though some success has recently been achieved inrice and corn. Hiei et al., The Plant Journal 6:271-282 (1994) and U.S.Pat. No. 5,591,616 issued Jan. 7, 1997. Several methods of planttransformation, collectively referred to as direct gene transfer, havebeen developed as an alternative to Agrobacterium-mediatedtransformation.

A generally applicable method of plant transformation ismicroprojectile-mediated transformation wherein DNA is carried on thesurface of microprojectiles measuring 1 to 4 micron. The expressionvector is introduced into plant tissues with a biolistic device thataccelerates the microprojectiles to speeds of 300 to 600 m/s which issufficient to penetrate plant cell walls and membranes. Sanford et al.,Part. Sci. Technol. 5:27 (1987), Sanford, J. C., Trends Biotech. 6:299(1988), Klein et al., BioTechnology 6:559-563 (1988), Sanford, J. C.,Physiol Plant 7:206 (1990), Klein et al., BioTechnology 10:268 (1992).In corn, several target tissues can be bombarded with DNA-coatedmicroprojectiles in order to produce transgenic plants, including, forexample, callus (Type I or Type II), immature embryos, and meristematictissue.

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang et al., BioTechnology 9:996 (1991). Alternatively,liposome and spheroplast fusion have been used to introduce expressionvectors into plants. Deshayes et al., EMBO J., 4:2731 (1985), Christouet al., Proc Natl. Acad. Sci. U.S.A. 84:3962 (1987). Direct uptake ofDNA into protoplasts using CaCl₂ precipitation, polyvinyl alcohol orpoly-L-ornithine has also been reported. Hain et al., Mol. Gen. Genet.199:161 (1985) and Draper et al., Plant Cell Physiol. 23:451 (1982).Electroporation of protoplasts and whole cells and tissues have alsobeen described. D'Halluin et al., Plant Cell 4:1495-1505 (1992) andSpencer et al., Plant Mol. Biol. 24:51-61 (1994).

Following transformation of corn target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods now well known in the art.

The foregoing methods for transformation would typically be used forproducing a transgenic inbred line. The transgenic inbred line couldthen be crossed, with another (non-transformed or transformed) inbredline, in order to produce a new transgenic inbred line. Alternatively, agenetic trait which has been engineered into a particular corn lineusing the foregoing transformation techniques could be moved intoanother line using traditional backcrossing techniques that are wellknown in the plant breeding arts. For example, a backcrossing approachcould be used to move an engineered trait from a public, non-eliteinbred line into an elite inbred line, or from an inbred line containinga foreign gene in its genome into an inbred line or lines which do notcontain that gene. As used herein, “crossing” can refer to a simple X byY cross, or the process of backcrossing, depending on the context.

When the term inbred corn plant is used in the context of the presentdisclosure, this also includes any inbred corn plant where one or moredesired traits have been introduced through backcrossing methods,whether such trait is a naturally occurring one or a transgenic one.Backcrossing methods can be used with the present disclosure to improveor introduce one or more characteristic into the inbred. The termbackcrossing as used herein refers to the repeated crossing of a hybridprogeny back to one of the parental corn plants for that inbred. Theparental corn plant which contributes the gene or the genes for thedesired characteristic is termed the nonrecurrent or donor parent. Thisterminology refers to the fact that the nonrecurrent parent is used onetime in the backcross protocol and therefore does not recur. Theparental corn plant to which the gene or genes from the nonrecurrentparent are transferred is known as the recurrent parent as it is usedfor several rounds in the backcrossing protocol (Fehr, 1987).

In a typical backcross protocol, the original inbred of interest(recurrent parent) is crossed to a second inbred (nonrecurrent parent)that carries the gene or genes of interest to be transferred. Theresulting progeny from this cross are then crossed again to therecurrent parent and the process is repeated until a corn plant isobtained wherein all the desired morphological and physiologicalcharacteristics of the recurrent parent are recovered in the convertedplant in addition to the gene or genes transferred from the nonrecurrentparent. It should be noted that some, one, two, three or more,self-pollination and growing of a population might be included betweentwo successive backcrosses. Indeed, an appropriate selection in thepopulation produced by the self-pollination, i.e., selection for thedesired trait and physiological and morphological characteristics of therecurrent parent might be equivalent to one, two or even threeadditional backcrosses in a continuous series without rigorousselection, saving time, money and effort for the breeder. The backcrossprocess could also be accelerated through a step of haploid inductiontogether by a molecular marker screening to identify the backcrossprogeny plants that have the closest genetic resemblance with therecurrent line, together with the gene or genes of interest to betransferred. A non-limiting example of such a protocol would be thefollowing: (a) the first generation F₁ produced by the cross of therecurrent parent A by the donor parent B is backcrossed to parent A, (b)selection is practiced for the plants having the desired trait of parentB, (c) selected plants are self-pollinated to produce a population ofplants where selection is practiced for the plants having the desiredtrait of parent B and the physiological and morphologicalcharacteristics of parent A, (d) the selected plants are backcrossedone, two, three, four, five or more times to parent A to produceselected backcross progeny plants comprising the desired trait of parentB and the physiological and morphological characteristics of parent A.Step (c) may or may not be repeated and included between the backcrossesof step d. Step (c) may or may not be followed by a step of haploidinduction followed by molecular marker screening.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute one or more trait(s) or characteristic(s) in theoriginal inbred. To accomplish this, a gene or genes of the recurrentinbred is modified or substituted with the desired gene or genes fromthe nonrecurrent parent, while retaining essentially all of the rest ofthe desired genetic, and therefore the desired physiological andmorphological, constitution of the original inbred. The choice of theparticular nonrecurrent parent will depend on the purpose of thebackcross; one of the major purposes is to add some commerciallydesirable, agronomically important trait(s) to the plant. The exactbackcrossing protocol will depend on the characteristic(s) or trait(s)being altered to determine an appropriate testing protocol. Althoughbackcrossing methods are simplified when the characteristic beingtransferred is a single gene and dominant allele, multiple genes andrecessive allele(s) may also be transferred and therefore, backcrossbreeding is by no means restricted to character(s) governed by one or afew genes. In fact the number of genes might be less important than theidentification of the character(s) in the segregating population. Inthis instance it may then be necessary to introduce a test of theprogeny to determine if the desired characteristic(s) has beensuccessfully transferred. Such tests encompass not only visualinspection and simple crossing, but also follow up of thecharacteristic(s) through genetically associated markers and molecularassisted breeding tools. For example, selection of progeny containingthe transferred trait is done by direct selection, visual inspection fora trait associated with a dominant allele, while the selection ofprogeny for a trait that is transferred via a recessive allele, such asthe waxy starch characteristic, require selfing the progeny to determinewhich plant carry the recessive allele(s).

Many single gene traits have been identified that are not regularlyselected for in the development of a new inbred but that can be improvedby backcrossing techniques. Single gene traits may or may not betransgenic, i.e., they may be naturally present in the nonrecurrentparent, examples of these traits include but are not limited to, malesterility, waxy starch, amylose starch, herbicide resistance, resistancefor bacterial, fungal, or viral disease, insect resistance, malefertility, water stress tolerance, enhanced nutritional quality,industrial usage, increased digestibility, yield stability and yieldenhancement. An example of this is the Rp1D gene which controlsresistance to rust fungus by preventing P. sorghi from producing spores.The Rp1D gene is usually preferred over the other Rp genes because it iswidely effective against all races of rust, but the emergence of newraces has lead to the use of other Rp genes comprising, for example, theRp1E, Rp1G, Rp1I, Rp1K or “compound” genes which combine two or more Rpgenes including Rp1GI, Rp1GDJ, etc. These genes are generally inheritedthrough the nucleus. Some known exceptions to this are the genes formale sterility, some of which are inherited cytoplasmically, but stillact as single gene traits. Several of these single gene traits aredescribed in U.S. Pat. Nos. 5,777,196; 5,948,957 and 5,969,212, thedisclosures of which are specifically hereby incorporated by reference.A exaemplary gene related to digestibility is known to one skilled inthe art, such as U.S. Pat. No. 8,143,482. Each of the referencesmentioned above is herein incorporated into reference by its entirety.

In 1981, the backcross method of breeding accounted for 17% of the totalbreeding effort for inbred line development in the United States,according to, Hallauer, A. R. et al., (1988) “Corn Breeding” in Corn andCorn Improvement, No. 18, pp. 463-481. The backcross breeding methodprovides a precise way of improving varieties that excel in a largenumber of attributes but are deficient in a few characteristics (Allard,1960, Principles of Plant Breeding, John Wiley & Sons, Inc.). The methodmakes use of a series of backcrosses to the variety to be improvedduring which the character or the characters in which improvement issought is maintained by selection. At the end of the backcrossing, thegene or genes being transferred unlike all other genes will beheterozygous. Selfing after the last backcross produces homozygosity forthis gene pair(s) and, coupled with selection, will result in a varietywith exactly the adaptation, yielding ability and qualitycharacteristics of the recurrent parent but superior to that parent inthe particular characteristic(s) for which the improvement program wasundertaken. Therefore, this method provides the plant breeder with ahigh degree of genetic control of his work.

Backcrossing is a powerful mechanism for achieving homozygosity and anypopulation obtained by backcrossing may rapidly converge on the genotypeof the recurrent parent. When backcrossing is made the basis of a plantbreeding program, the genotype of the recurrent parent will be modifiedwith regards to genes being transferred, which are maintained in thepopulation by selection.

Examples of successful backcrosses are the transfer of stem rustresistance from “Hope” wheat to “Bart” wheat and the transfer of buntresistance to “Bart” wheat to create “Bart 38” which has resistance toboth stem rust and bunt. Also highlighted by Allard is the successfultransfer of mildew, leaf spot and wilt resistances in “CaliforniaCommon” alfalfa to create “Caliverde.” This new “Caliverde” varietyproduced through the backcross process is indistinguishable from“California Common” except for its resistance to the three nameddiseases.

One of the advantages of the backcross method is that the breedingprogram can be carried out in almost every environment that will allowthe development of the character being transferred. Another advantage ofthe backcross method is that more than one character or trait can betransferred, either through several backcrosses or through the use oftransformation and then backcrossing.

The backcross technique is not only desirable when breeding for diseaseresistance but also for the adjustment of morphological characters,color characteristics and simply inherited quantitative characters suchas earliness, plant height and seed size and shape. In this regard, amedium grain type variety, “Calady,” has been produced by Jones andDavis. In dealing with quantitative characteristics, they selected thedonor parent with the view of sacrificing some of the intensity of thecharacter for which it was chosen, i.e., grain size. “Lady Wright,” along grain variety was used as the donor parent and “Coloro,” a shortgrain one as the recurrent parent. After four backcrosses, the mediumgrain type variety “Calady” was produced.

DEPOSIT INFORMATION

A deposit of the inbred corn line of this disclosure is maintained byAgReliant Genetics, LLC, 1107 Baseline Road, Esmond, IL, 60129.AgReliant maintains the seed deposit on behalf of KWS SAAT SE & Co.KGaA. In addition, a sample of the inbred corn seed of this disclosurehas been deposited with the American Type Culture Collection, 10801University Boulevard, Manassas, Virginia 20110. The deposit for theinbred corn line KW4ER1502 will be made on ______.

To satisfy the enablement requirements of 35 U.S.C. 112, and to certifythat the deposit of the isolated strain (i.e., corn inbred) of thepresent disclosure meets the criteria set forth in 37 CFR 1.801-1.809and Manual of Patent Examining Procedure (MPEP) 2402-2411.05, Applicantshereby make the following statements regarding the deposited inbred cornline KW4ER1502 (deposited as ATCC Accession No. PTA-______):

If the deposit is made under the terms of the Budapest Treaty, theinstant disclosure will be irrevocably and without restriction releasedto the public upon the granting of a patent.

If the deposit is made not under the terms of the Budapest Treaty,Applicant(s) provides assurance of compliance by following statements:

-   -   1. During the pendency of this application, access to the        disclosure will be afforded to the Commissioner upon request;    -   2. All restrictions on availability to the public will be        irrevocably removed upon granting of the patent under conditions        specified in 37 CFR 1.808;    -   3. The deposit will be maintained in a public repository for a        period of 30 years or 5 years after the last request or for the        effective life of the patent, whichever is longer;    -   4. A test of the viability of the biological material at the        time of deposit will be conducted by the public depository under        37 CFR 1.807; and    -   5. The deposit will be replaced if it should ever become        unavailable.

Access to this deposit will be available during the pendency of thisapplication to persons determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. § 1.14 and 35 U.S.C. §122. Upon granting of any claims in this application, all restrictionson the availability to the public of the variety will be irrevocablyremoved by affording access to a deposit of at least 625 seeds of thesame variety with the ATCC.

INDUSTRIAL APPLICABILITY

Corn is used as human food, livestock feed, and as raw material inindustry. The food uses of corn, in addition to human consumption ofcorn kernels, include both products of dry- and wet-milling industries.The principal products of corn dry-milling are grits, meal and flour.Corn meal is flour ground to fine, medium, and coarse consistencies fromdried corn. In the United States, the finely ground corn meal is alsoreferred to as corn flour. However, the term “corn flour” denotes cornstarch in the United Kingdom. Corn meal has a long shelf life and isused to produce an assortment of products, including but not limited totortillas, taco shells, bread, cereal and muffins.

The corn wet-milling industry can provide corn starch, corn syrups, cornsweeteners and dextrose for food use. Corn syrup is used in foods tosoften texture, add volume, prevent crystallization of sugar and enhanceflavor. Corn syrup is distinct from high-fructose corn syrup (HFCS),which is created when corn syrup undergoes enzymatic processing,producing a sweeter compound that contains higher levels of fructose.

Corn oil is recovered from corn germ, which is a by-product of both dry-and wet-milling industries. Corn oil which is high in mono and polyunsaturated fats, is used for reducing fat and trans fat in numerousfood products.

Corn, including both grain and non-grain portions of the plant, is alsoused extensively as livestock feed, primarily for beef cattle, dairycattle, hogs and poultry.

Industrial uses of corn include production of ethanol, corn starch inthe wet-milling industry and corn flour in the dry-milling industry.Corn ethanol is ethanol produced from corn as a biomass throughindustrial fermentation, chemical processing and distillation. Corn isthe main feedstock used for producing ethanol fuel in the United States.The industrial applications of corn starch and flour are based onfunctional properties, such as viscosity, film formation, adhesiveproperties, and ability to suspend particles. Corn starch and flour alsohave application in the paper and textile industries. Other industrialuses include applications in adhesives, building materials, foundrybinders, laundry starches, explosives, oil-well muds and other miningapplications.

Plant parts other than the grain of corn are also used in industry, forexample, stalks and husks are made into paper and wallboard and cobs areused for fuel and to make charcoal.

The seed of inbred corn line KW4ER1502, the plant produced from theinbred seed, the hybrid corn plant produced from the crossing of theinbred, hybrid seed, and various parts of the hybrid corn plant andtransgenic versions of the foregoing, can be utilized for human food,livestock feed, and as a raw material in industry.

Tables of Field Test Trials

In the tables that follow, exemplary traits and characteristics ofhybrid combinations having inbred corn line KW4ER1502 as a parental lineare given compared to other hybrids. The data collected are presentedfor key characteristics and traits. The field tests are experimentaltrials and have been made at numerous locations, with one or tworeplications per location under supervision of the applicant.Information about these experimental hybrids as compared to the checkhybrids is presented.

Field Test Trials:

Information for each pedigree includes:

-   -   1. Mean yield of the hybrid (YLD) across all locations        (bushels/acre) is shown in column 2.    -   2. A mean for the percentage moisture (Mst %) for the hybrid        across all locations is shown in column 3.    -   3. A mean of the yield divided by the percentage moisture (Y/M)        for the hybrid across all locations is shown in column 4.    -   4. Test weight (TWgt) is the grain density as measured in pounds        per bushel and is shown in column 5.    -   5. A mean of the percentage of plants with stalk lodging (SL %)        across all locations is shown in column 6.    -   6. A mean of the percentage of plants with root lodging (RL %)        across all locations is shown in column 7.    -   7. Ear Height (EHT) is a physical measurement taken from the        ground level to the node of attachment for the upper ear. It is        expressed to the nearest centimeter and is shown in column 8.    -   8. Plant Height (PLHT) is a physical measurement taken from the        ground level to the flag leaf. It is expressed to the nearest        centimeter and is shown in column 9.    -   9. Harvest Appearance (HA n) is a rating made by a trained        person on the date of harvest. Harvest appearance is the rater's        impression of the hybrid based on, but not limited to, a        combination of factors to include plant intactness, tissue        health appearance and ease of harvest as it relates to stalk        lodging and root lodging. A scale of 1=Lowest to 9=Highest/Most        Desirable is used and is listed in column 10.

TABLE 2 Comparative Data for One Experimental Hybrid Having KW4ER1502 asOne Inbred Parent Overall comparisons: Year 1 field trials, 6 repsPedigree YLD Mst % Y/M TWgt SL % RL % EHT PLHT HA n Control Hybird 2192.0 23.0 8.4 53.7 0.4 0.4 240 105 5.4 Experimental Hybrid 1 197.8 23.08.6 51.8 0.1 2.1 240 120 5.6 Control Hybird 4 189.2 24.1 7.9 52.6 0.00.8 250 110 5.4 Control Hybird 1 196.5 24.2 8.1 53.0 0.0 0.8 250 115 5.4Control Hybird 3 203.3 24.6 8.3 53.7 0.0 0.0 250 110 5.9

TABLE 3 Comparative Data for One Experimental Hybrid Having KW4ER1502 asOne Inbred Parent Overall comparisons: Year 2 field trials, 9 repsPedigree YLD Mst % Y/M TWgt SL % RL % EHT PLHT HA n Control Hybird 5230.4 17.6 13.1 56.3 1.0 0.0 5.8 Experimental Hybrid 2 238.5 18.0 13.256.5 0.0 0.0 6.9 Control Hybird 6 230.2 18.0 12.8 56.0 0.0 0.0 5.6Control Hybird 2 239.5 19.4 12.4 56.1 0.0 0.0 6.1 Control Hybird 1 245.019.5 12.5 56.1 0.0 0.0 6.0 Control Hybird 3 241.8 19.8 12.2 56.3 0.0 2.86.3

TABLE 4 Comparative Data for One Experimental Hybrid Having KW4ER1502 asOne Inbred Parent Overall comparisons: Year 2 field trials, 13 repsPedigree YLD Mst % Y/M TWgt SL % RL % EHT PLHT HA n Control Hybird 5229.8 17.4 13.2 56.3 0.0 0.8 245 120 5.4 Control Hybird 6 241.7 17.813.6 56.8 0.0 5.8 243 112 5.8 Control Hybird 1 243.4 18.9 12.9 56.5 0.01.9 253 122 6.0 Control Hybird 2 238.4 19.0 12.6 56.6 0.0 1.5 247 1156.3 Experimental Hybrid 3 230.6 19.2 12.0 55.6 0.0 0.0 247 120 6.4Control Hybird 3 243.7 19.4 12.5 56.6 0.0 0.4 250 122 6.6

TABLE 5 Comparative Data for One Experimental Hybrid Having KW4ER1502 asOne Inbred Parent Overall comparisons: Year 2 field trials, 32 repsPedigree YLD Mst % Y/M TWgt SL % RL % EHT PLHT HA n Control Hybird 5230.4 17.0 13.5 56.4 0.0 1.1 254 118 5.8 Control Hybird 6 241.6 17.813.6 56.5 0.0 1.0 259 121 5.6 Experimental Hybrid 4 243.1 18.2 13.4 56.41.3 1.3 259 124 6.2 Control Hybird 1 246.1 18.6 13.2 56.7 0.0 1.8 260118 6.0 Control Hybird 2 242.8 18.8 12.9 56.7 0.0 0.2 244 117 6.2Control Hybird 3 245.1 19.2 12.8 56.6 0.0 0.3 255 120 6.2

TABLE 6 Comparative Data for One Experimental Hybrid Having KW4ER1502 asOne Inbred Parent Overall comparisons: Year 3 field trials, 14 repsPedigree YLD Mst % Y/M TWgt SL % RL % EHT PLHT HA n Control Hybird 6244.4 16.5 14.8 57.5 0.2 4.3 256 114 6.0 Control Hybird 2 241.9 17.214.1 57.9 0.0 2.9 256 112 6.3 Control Hybird 1 248.6 17.3 14.4 57.7 0.34.6 263 110 6.2 Control Hybird 3 248.4 17.6 14.1 58.1 0.3 2.8 254 1086.1 Experimental Hybrid 5 254.6 18.1 14.1 58.0 0.0 0.4 274 115 7.2Control Hybird 7 264.9 18.3 14.4 57.0 0.0 4.1 271 117 6.9

The foregoing detailed description has been given for clearness ofunderstanding only and no unnecessary limitations should be understoodthere from as modifications will be obvious to those skilled in the art.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions and sub-combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions and sub-combinations as are within their truespirit and scope.

Unless defined otherwise, all technical and scientific terms herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this disclosure belongs. Although any methods andmaterials, similar or equivalent to those described herein, can be usedin the practice or testing of the present disclosure, the preferredmethods and materials are described herein. All publications, patents,and patent publications cited are incorporated by reference herein intheir entirety for all purposes.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present disclosure isnot entitled to antedate such publication by virtue of prior disclosure.

While the disclosure has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the disclosure following, in general, theprinciples of the disclosure and including such departures from thepresent disclosure as come within known or customary practice within theart to which the disclosure pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

What is claimed is:
 1. A seed of inbred corn line designated KW4ER1502,wherein a representative sample of seed of said line was deposited underATCC Accession No. PTA-______.
 2. A corn plant, or a part thereof,produced by growing the seed of claim
 1. 3. A corn plant, or a partthereof, having all of the physiological and morphologicalcharacteristics of inbred corn line KW4ER1502, wherein a representativesample of seed of said line was deposited under ATCC Accession No.PTA-______.
 4. A tissue culture of cells produced from the plant or thepart of claim
 2. 5. A corn plant regenerated from the tissue culture ofclaim 4, wherein the regenerated plant has all of the morphological andphysiological characteristics of inbred corn line KW4ER1502, wherein arepresentative sample of seed of said line was deposited under ATCCAccession No. PTA-______.
 6. A method for producing a hybrid corn seed,the method comprising: crossing the plant of claim 2 with a differentcorn plant, and harvesting the resultant hybrid corn seed.
 7. A hybridcorn seed produced by the method of claim
 6. 8. An F1 hybrid corn plant,or a part thereof, produced by growing the seed of claim
 7. 9. A methodfor producing inbred corn line KW4ER1502, wherein a representativesample of seed of said line was deposited under ATCC Accession No.PTA-______, the method comprising: (a) planting a collection of seedscomprising seed of a hybrid, one of whose parents is inbred corn lineKW4ER1502, the collection also comprising seed of the inbred corn line;(b) growing plants from the collection of seeds; (c) identifying theplants having all of the physiological and morphological characteristicsof inbred corn line KW4ER1502 as inbred parent plants; (d) controllingpollination of the inbred parent plants in a manner that preserves thehomozygosity of the inbred parent plants; and (e) harvesting theresultant seed and thereby producing an inbred corn line having all ofthe physiological and morphological characteristics of inbred corn lineKW4ER1502.
 10. The method of claim 9, wherein step (c) comprisesidentifying plants with decreased vigor compared to the other plantsgrown from the collection of seeds.
 11. A method for producing a cornplant that contains in its genetic material one or more transgenes, themethod comprising: crossing the plant of claim 2 with either a secondplant of another corn line that contains a transgene or a transformedcorn plant of the inbred corn line KW4ER1502, so that the geneticmaterial of a progeny plant that results from the cross contains thetransgene(s) operably linked to a regulatory element, wherein thetransgene confers a trait selected from the group consisting of malesterility, male fertility, herbicide resistance, insect resistance,disease resistance, water stress tolerance, and increased digestibility.12. A corn plant, or a part thereof, produced by the method of claim 11.13. The corn plant of claim 12, wherein the transgene confers resistanceto an herbicide selected from the group consisting of imidazolinone,sulfonylurea, glyphosate, glufosinate, L-phosphinothricin, triazine andbenzonitrile.
 14. The corn plant of claim 12, wherein the transgeneencodes a Bacillus thuringiensis protein.
 15. The corn plant of claim12, wherein the transgene confers disease resistance.
 16. The corn plantof claim 12, wherein the transgene confers water stress tolerance. 17.The corn plant of claim 12, wherein the transgene confers increaseddigestibility.
 18. A method for producing a hybrid corn seed, the methodcomprising: crossing the plant of claim 12 with a different corn plant,and harvesting the resultant hybrid corn seed.
 19. A method of producinga corn plant with increased waxy starch or increased amylose starch, themethod comprising: transforming the corn plant of claim 2 with atransgene that modifies waxy starch or amylose starch metabolism,thereby producing the corn plant with increased waxy starch or amylosestarch metabolism.
 20. A corn plant produced by the method of claim 19.21. A method of introducing one or more desired traits into inbred cornline KW4ER1502, the method comprising: (a) crossing inbred corn lineKW4ER1502 plants grown from inbred corn line KW4ER1502 seeds, wherein arepresentative sample of seed of the line was deposited under ATCCAccession No. PTA-______, with plants of another corn line that compriseone or more desired traits to produce progeny plants, wherein the one ormore desired traits are selected from the group consisting of malesterility, male fertility, herbicide resistance, insect resistance,disease resistance, waxy starch, water stress tolerance, increasedamylose starch and increased digestibility; (b) selecting progeny plantsthat have the one or more desired traits to produce selected progenyplants; (c) crossing the selected progeny plants with the inbred cornline KW4ER1502 plants to produce backcross progeny plants; (d) selectingfor backcross progeny plants that have the one or more desired traits toproduce selected backcross progeny plants; and (e) repeating steps (c)and (d) one or more times in succession to produce selected second orhigher backcross progeny plants that comprise the one or more desiredtraits and all of the physiological and morphological characteristics ofinbred corn line KW4ER1502.
 22. A corn plant produced by the method ofclaim 21, wherein the plant has the one or more desired traits and allof the physiological and morphological characteristics of inbred cornline KW4ER1502.
 23. A method for producing inbred corn line KW4ER1502seed, wherein a representative sample of seed of said line was depositedunder ATCC Accession No. PTA-______, the method comprising: crossing afirst inbred corn plant with a second inbred corn plant, and harvestingthe resultant corn seed, wherein both the first and second inbred cornplants are the plant of claim
 2. 24. A method for producing inbred cornline KW4ER1502 seed, wherein a representative sample of seed of saidline was deposited under ATCC Accession No. PTA-______, the methodcomprising: (a) planting the seed of claim 1; (b) growing a plant fromsaid seed; (c) controlling pollination in a manner that the pollenproduced by the grown plant pollinates the ovules produced by the grownplant; and (d) harvesting the resultant seed and thereby producing aninbred corn line having all of the physiological and morphologicalcharacteristics of inbred corn line KW4ER1502.
 25. A method forproducing a corn seed that contains in its genetic material one or moretransgenes, wherein the method comprises crossing the corn plant ofclaim 2 with either a second plant of another corn line which containsone or more transgenes or a transformed corn plant of the inbred cornline KW4ER1502, wherein the transgene(s) is operably linked to aregulatory element and wherein the transgene confers a trait selectedfrom the group consisting of male sterility, male fertility, herbicideresistance, insect resistance, disease resistance, water stresstolerance, and increased digestibility, and harvesting the resultantseed.
 26. A corn seed, or a part thereof, produced by the method ofclaim
 25. 27. A method for producing a hybrid corn seed, the methodcomprising: crossing the plant of claim 22 with a different corn plant,and harvesting the resultant hybrid corn seed.
 28. An F1 hybrid cornseed produced by the method of claim
 27. 29. A method of producing acorn product, the method comprising the step of milling the seed ofclaim 1, thereby producing the corn product.
 30. The method of claim 29,wherein the corn product is selected from the group consisting of cornmeal, corn flour, corn starch, corn syrup, corn sweetener and corn oil.31. A method of producing a corn product, the method comprising the stepof milling the hybrid seed of claim 7, thereby producing the cornproduct.
 32. The method of claim 31, wherein the corn product isselected from the group consisting of corn meal, corn flour, cornstarch, corn syrup, corn sweetener and corn oil.